Activation of AMPK Enhances Neutrophil Chemotaxis and Bacterial Killing
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An inability of neutrophils to eliminate invading microorganisms is frequently associated with severe infection and may contribute to the high mortality rates associated with sepsis. In the present studies, we examined whether metformin and other 5′ adenosine monophosphate-activated protein kinase (AMPK) activators affect neutrophil motility, phagocytosis and bacterial killing. We found that activation of AMPK enhanced neutrophil chemotaxis in vitro and in vivo, and also counteracted the inhibition of chemotaxis induced by exposure of neutrophils to lipopolysaccharide (LPS). In contrast, small interfering RNA (siRNA)-mediated knockdown of AMPKα1 or blockade of AMPK activation through treatment of neutrophils with the AMPK inhibitor compound C diminished neutrophil chemotaxis. In addition to their effects on chemotaxis, treatment of neutrophils with metformin or aminoimidazole carboxamide ribonucleotide (AICAR) improved phagocytosis and bacterial killing, including more efficient eradication of bacteria in a mouse model of peritonitis-induced sepsis. Immunocytochemistry showed that, in contrast to LPS, metformin or AICAR induced robust actin polymerization and distinct formation of neutrophil leading edges. Although LPS diminished AMPK phosphorylation, metformin or AICAR was able to partially decrease the effects of LPS/toll-like receptor 4 (TLR4) engagement on downstream signaling events, particularly LPS-induced IκBα degradation. The IκB kinase (IKK) inhibitor PS-1145 diminished IκBα degradation and also prevented LPS-induced inhibition of chemotaxis. These results suggest that AMPK activation with clinically approved agents, such as metformin, may facilitate bacterial eradication in sepsis and other inflammatory conditions associated with inhibition of neutrophil activation and chemotaxis. Online address: https://doi.org/www.molmed.org
Neutrophils are an essential component of the innate immune system, with a primary role in the clearance of extracellular pathogens (1). Both localization and neutralization of microorganisms are important neutrophil functions that are orchestrated by specific inflammatory mediators released from the site of infection (2,3). In particular, chemoattractants and chemokine gradients are major neutrophil guidance signals, whereas the migration of neutrophils from the vasculature to inflammatory sites is mediated by adhesion proteins, including P- and E-selectins and integrin ligands such as vascular cell adhesion molecule 1 (VCAM-1), intracellular adhesion molecule 1 (ICAM-1) and ICAM-2 on endothelium (2,4). Several mechanisms are involved in bacterial killing by neutrophils. For example, release of antimicrobial peptides, generation of reactive nitrogen and oxygen species (ROS/RNS), as well as production of hypochlorous acid by myeloperoxidase, are utilized by neutrophils to kill invading microorganisms (5,6). In addition to phagocytosis and killing internalized microorganisms, neutrophils also can release DNA and DNA-associated proteins to form extracellular traps to prevent bacterial dissemination (7,8).
Although killing of microorganisms is apparently a beneficial function of neutrophil activation, exaggerated activation of neutrophils can result in collateral damage to tissues and also contribute to the development of organ failure in sepsis (9,10). However, impairment of neutrophil activation also can lead to serious complications in infected patients. In particular, diminished neutrophil activation or decreased neutrophil numbers are associated with a high mortality rate in sepsis (11). Neutrophil dysfunction also commonly occurs with bacterial pneumonia in critically ill patients after trauma and hemorrhage (12, 13, 14, 15).
Beside the central roles that NADPH-oxidase and ROS occupy in killing microorganisms, enhanced propagation and dissemination of bacteria are associated with loss of neutrophil ability to reach the site of infection (16,17). In sepsis, such alterations in neutrophil function are a result of disrupted chemokine signaling and expression of adhesion molecules (16, 17, 18, 19, 20, 21). For example, the appearance of detectable levels of the bacterial products lipopolysaccharide (LPS) or lipoteichoic acid in the circulation of severely infected patients is associated with diminished neutrophil chemotaxis, including neutrophil response to interleukin 8 (IL-8), macrophage inflammatory protein 2 (MIP-2), or keratinocyte-derived chemokine (KC). In spite of progress in understanding mechanisms responsible for the inhibition of neutrophil chemotaxis, pharmacologic approaches to prevent or restore neutrophil chemotaxis and bacterial eradication are not available.
Metformin is commonly used in patients with non-insulin-dependent diabetes mellitus to lower blood glucose concentrations and to improve insulin sensitivity (22). Metformin has been shown to prevent cardiovascular complications associated with diabetes and obesity through mechanisms that presumably involve inhibition of adipose tissue lipolysis, reduction of circulating levels of free fatty acids, and inhibition of low density lipoprotein production (23,24). Although metformin has a broad spectrum of effects, inhibition of mitochondrial complex I and activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) appear to be major mechanisms of its action (25,26). Recent studies have shown that besides their ability to regulate cellular metabolism (27), metformin and other AMPK activators can decrease the severity of organ injury in acute inflammatory states (28), including LPS-induced liver injury or acute lung injury (29,30). Metformin, berberine or aminoimidazole carboxamide ribonucleotide (AICAR) were all shown to diminish activation of the tolllike receptor 4 (TLR4)/NF-κB signaling cascade, as well as to release of proin-flammatory mediators of neutrophils and macrophages, and also to enhance endothelial integrity in vitro and in models for sepsis (31, 32, 33, 34, 35, 36). In addition, metformin enhances host defense mechanisms by facilitating the chemotaxis and maturation of T cells (37,38). Besides its antiinflammatory actions, activated AMPK was recently shown to increase the phagocytic ability of macrophages (39). Although these studies revealed a beneficial effect of metformin in acute inflammatory conditions, there is little information concerning the ability of metformin or other AMPK activators to alter primary innate immune responses, and particularly bacterial eradication. In this study, we examined the hypothesis that AMPK activation may affect fundamental neutrophil functions, including chemotaxis and bacterial killing.
Materials and Methods
Male C57BL/6 mice were purchased from the National Cancer Institute-Frederick (Frederick, MD, USA). Male mice, 8 to 10 wks of age, were used for experiments. The mice were kept on a 12-h light:dark cycle with free access to food and water. All experiments were conducted in accordance with protocols approved by the University of Alabama at Birmingham Animal Care and Use Committee.
Reagents and Antibodies
W-peptide was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). Metformin, berberine and IκB kinase (IKK) inhibitor PS-1145 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Compound C and rapamycin were purchased from Calbiochem (La Jolla, CA, USA). Antibodies for phospho-Thr172-AMPK, total AMPK, phospho-Ser240/244-rpS6, total rpS6, 4E-BP1, and IκBα were purchased from Cell Signaling Technology (Danvers, MA, USA). β-actin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Custom antibody mixtures and negative selection columns for neutrophil isolation were obtained from Stem Cell Technologies (Vancouver, BC, Canada). Fluorescein isothiocyanate (FITC)-labeled E. coli, S. aureus and Alexa Fluor 594-conjugated phalloidin were from Invitrogen/Life Technologies (Carlsbad, CA, USA). Mounting oil solution containing DAPI was from Vector laboratories (Burlingame, CA, USA). The µ-slide for chemotaxis assay was obtained from Ibidi (Mt. Prospect, IL, USA); and transmigration chambers that we used are available from BD Biosciences (San Jose, CA, USA).
Neutrophil Isolation and Culture
Bone marrow neutrophils were isolated as described previously (31,40). Neutrophil purity was consistently greater than 97%, as determined by Wright-Giemsa-stained cytospin preparations. Neutrophils were cultured in RPMI 1640 medium containing 0.5% or 5% fetal bovine serum (FBS) and treated as indicated in the figure legends. Neutrophil viability under experimental conditions was determined using trypan blue staining and was consistently greater than 95%.
HL-60 Cell Culture and Differentiation
The HL-60 cell line was obtained from American Type Culture Collection and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL), streptomycin (25 µg/mL) and L-glutamine (1 mmol/L). To obtain differentiated cells, HL-60 cells were cultured in medium containing dimethyl sulfoxide (DMSO) (1.3%) for four consecutive days (41). Differentiated cells were then used for small interfering RNA (siRNA) treatment and IL-8 mediated chemotaxis.
siRNA Knockdown of AMPKα1
Differentiated HL-60 cells were incubated with specific siRNA (1 µmol/L) to AMPKα1, as described previously (41). Briefly, cells (3.5 × 106/well) in 12-well plates were incubated in Accell medium (serum free) containing siRNA (1 µmol/L) to AMPKα1 for 72 h. During incubation with siRNA, the cell culture medium was supplemented with 1.3% DMSO to maintain differentiation of the HL-60 cells. The cells were then subjected to Western blot analysis of AMPK or transwell migration assay.
Transwell Migration Assay
Transwell migration assay was performed using 24-well cell plate BD Falcon cell culture inserts (Translucent PET Membrane, BD Biosciences). Briefly, bone marrow neutrophils (106 cells/well) or differentiated HL-60 cells in 300 µL of RPMI 1640 medium (5% FBS) were added to the upper reservoir, whereas W-peptide or IL-8 in culture medium (800 µL) was placed in the lower reservoir of transmigration chamber. In all experiments, chemotaxis was determined after neutrophils or HL-60 cells were allowed to migrate for 60 min at 37°C followed by imaging the cells in the lower reservoir (41). Each condition was tested three or more times.
Measurement of Cell Velocity
Bone marrow neutrophils pretreated as described in figure legends and then loaded into the µ-slide for chemotaxis assay (Ibidi). Cell migration was initiated by inclusion of W-peptide (50 nmol/L) to create a concentration gradient. Migration was recorded by imaging cells with 1 min intervals for a total of 60 min and then distance and movement direction of individual cells were plotted to calculate speed and velocity.
Measurement Neutrophil Chemotaxis In Vivo
Mice were subjected to application of metformin (125 mg/kg of body weight; intraperitoneal [IP]) for 12 h and 2 h before IP injections of W-peptide (0.43 mg/kg). In additional experiments, mice were treated with compound C (3 mg/kg, IP) or vehicle (saline) for 2 h prior to application of W-peptide. After 6 h, mice were euthanized and peritoneal lavages obtained using 10 mL of RPMI 1640 medium (without serum).
Phagocytosis of fluorescent labeled E. coli or S. aureus by neutrophils was performed as described previously (39). In brief, phagocytosis of fluorescently labeled bacteria by neutrophils pretreated with or without metformin (500 µmol/L, 2.5 h) was determined by adding tenfold excess of E. coli or S. aureus to the cells. To measure internalization of bacteria, fluorescent E. coli or S. aureus were incubated for 15 min at 37°C, and cells were then washed three times in ice-cold PBS. Next, cells were incubated with or without trypan blue solution (0.2% trypan blue, 20 mmol/L citrate, and 150 mmol/L NaCl, pH 4.5) for 1 min, then centrifuged, and the cell pellet was resuspended in PBS, and the amount of fluorescence was measured using flow cytometry.
In Vitro Killing-Activity Assay
Neutrophils (2 × 106 cells/mL) were incubated with ampicillin-resistant E. coli (2 x 107/mL) in RPMI medium without serum for 90 min at 37°C. Next, 20 µL of cell/bacterial suspension was incubated with 480 µL Triton X-100 (0.1%) for 10 min to lyse neutrophils. Serial dilutions were then plated on agar plates with ampicillin and incubated overnight at 37°C. The number of bacterial colonies on agar plates was determined using colony counter software (Bio-Rad, Hercules, CA, USA).
The efficiency of bacterial eradication in vivo was performed as described previously (42,43). Mice were subjected to administration of metformin (125 mg/kg of body weight; IP) for 12 h, compound C (3 mg/kg of body weight; IP) or vehicle (PBS) for 2 h before IP injection of ampicillin-resistant E. coli (2 × 108). After 6 h, mice were euthanized and peritoneal lavages obtained using 10 mL RPMI 1640 medium without serum. The number of surviving bacteria was determined by incubation of 95 µL of peritoneal lavages with 5 µL of Triton X-100 (1%) for 10 min to lyse cells, and then serial dilutions were placed on agar plates with ampicillin and incubated overnight at 37°C. Bacterial colonies were counted using colony counter software (Bio-Rad).
Imaging Actin in Neutrophils
Neutrophils were incubated with 4% paraformaldehyde in PBS for 30 min at room temperature then washed with PBS and permeabilized with 0.1% Triton X-100/PBS for 4 min. The cells were then incubated with 3% BSA in PBS for 1 h, followed by the addition of Alexa Fluor 594-conjugated phalloidin (25 µL/mL) for 20 min at room temperature. After the cells were washed with PBS, they were mounted with emulsion oil solution containing DAPI to visualize nuclei. Confocal microscopy was performed as described previously, using a Leica DMIRBE inverted epifluorescence/Nomarski microscope (Leica Microsystems, Wetzlar, Germany) outfitted with Leica TCS NT laser confocal optics (40).
Western Blot Analysis
Western Blot analysis was performed as described previously (44,45). Briefly, cell lysates of murine bone marrow neutrophils (3.5 × 106/well) were prepared using lysis buffer containing Tris pH 7.4 (50 mmol/L), NaCl (150 mmol/L), NP-40 (0.5%, vol/vol), EDTA (1 mmol/L), EGTA (1 mmol/L), okadaic acid (1 nmol/L) and protease inhibitors. Cell lysates were sonicated and then centrifuged at 10,000g for 15 min at 4°C to remove insoluble material. The protein concentration in the supernatants was determined using the Bradford reagent (BioRad) with BSA as a standard. Samples were mixed with Laemmli sample buffer and boiled for 15 min. Equal amounts of proteins were resolved by 8% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon P; Millipore, Billerica, MA, USA). The membranes were probed with specific antibodies as described in the figure legends followed by detection with HRP-conjugated goat anti-rabbit IgG. Bands were visualized by enhanced chemiluminescence (Super Signal; Pierce Biotechnology, Rockford, IL, USA) and quantified by AlphaEaseFC software (Alpha Innotech, San Leandro, CA, USA). Each experiment was carried out two or more times using cell populations obtained from separate groups of mice.
Statistical significance was determined by the Wilcoxon rank sum test (independent two-group Mann-Whitney U test) as well as Student t test for comparisons between two groups. Multigroup comparisons were performed using one-way analysis of variance (ANOVA) with Tukey post hoc test. A value of P less than 0.05 was considered significant. Analyses were performed on SPSS version 16.0 (IBM, Armonk, NY, USA) for Windows (Microsoft Corporation, Redmond, WA, USA).
Effects of AMPK Inhibition or Activation on Neutrophil Chemotaxis
AMPK Activation Prevents LPS-Induced Inhibition of Chemotaxis
Given our in vitro results showing that AMPK activation modulated neutrophil chemotaxis, we determined if activation or inhibition of AMPK can affect neutrophil chemotaxis in vivo. To examine this possibility, control mice or mice treated with metformin or compound C were then given an IP injection of W-peptide and the number of neutrophils migrating into the peritoneum was measured 5 h later (Figure 4D). As shown in Figure 4E, treatment with metformin before application of W-peptide was associated with an increased number of peritoneal neutrophils as compared with treatment with W-peptide alone. In contrast to metformin, administration of compound C diminished W-peptide-induced peritoneal accumulation of neutrophils.
AMPK Activation Facilitates Neutrophil-Dependent Bacterial Uptake and Killing
Previous studies have shown that signaling cascades downstream of AMPK activates regulation of mTORC1. Therefore, in additional experiments, neutrophils were treated with the specific mTOR inhibitor rapamycin. However, rapamycin exposure did not affect the ability of neutrophils to eradicate bacteria (see Figures 5C, D).
AMPK Activation Enhances Bacterial Killing in Peritonitis-Induced Sepsis
Considering our in vitro results that AMPK activation with metformin increased neutrophil chemotaxis, as well as enhanced uptake and killing bacteria in vitro, we determined if metformin treatment can improve bacterial clearance in vivo. This possibility was examined using a murine model for peritonitis-induced sepsis (42,43). As shown in Figures 5E and 5F, there were significant decreases in the numbers of viable bacteria recovered from mice that received metformin as compared with controls. Of note, the number of peritoneal neutrophils was found to be increased, compared with the control group (Figure 5G).
AMPK Activation Stimulates Actin Rearrangement, Neutrophil Leading Edge Formation, and Diminishes LPS/TLR4-Mediated Inhibition of Neutrophil Chemotaxis
In the present studies, we found that activation of AMPK enhanced neutrophil chemotaxis and bacterial uptake, both essential components of bacterial killing. Recent studies, including results obtained from our laboratory (39), have shown that metformin or other AMPK activators enhance cell mobility and also phagocytosis. For example, AMPK activation was associated with enhanced T cell chemotaxis or migration of epithelial cells (38,50). AMPK activation has also been shown to enhance the phagocytic ability of macrophages, including uptake of bacteria, synthetic beads or apoptotic cells (39). These results are consistent with an ability of activated AMPK to facilitate microbial eradication through mechanisms that involve enhancement of neutrophil chemotaxis and/or bacterial uptake. Consistent with these previously reported findings, our present in vivo results showed that metformin effectively improved bacterial killing in vivo.
Severe sepsis is characterized by alterations in immunologic and host defense functions that include downregulation of neutrophil chemotaxis and phagocytosis (2,16,17,51,52). Because AMPK activation in metformin-treated neutrophils prevented LPS-mediated inhibition of chemotaxis, it is possible that metformin treatment may preserve neutrophil chemotaxis in the setting of sepsis and facilitate the ability of the host to clear invading pathogens. Our results showed that metformin can reverse the inhibitory actions of LPS on chemotaxis, and also prevent LPS-induced degradation of IκBα. Of note, exposure of neutrophils to the IKK inhibitor PS-1145 (53,54) also diminished IκBα degradation and prevented inhibition of neutrophil chemotaxis by LPS, suggesting that the effects of AMPK activation in this setting may also be due to its ability to inhibit IκBα degradation. Although previous studies have shown that metformin and other AMPK activators are capable of inhibiting mTORC1 function (55,56), in the present experiments, exposure of neutrophils to metformin did not prevent LPS-dependent activation of mTOR. These results suggest that AMPK activation enhances neutrophil chemotaxis through mechanisms that involve suppression of TLR4-associated signaling pathways other than those involving mTOR. Although our results suggest that AMPK activation has beneficial effects on neutrophil function related to microbial clearance, it will be important to determine how AMPK activation affects such functions in additional cell populations, and also if AMPK activation can restore the diminished monocyte and T cell responses frequently found in sepsis (57).
Previous studies and results obtained from our laboratory have described antiinflammatory effects mediated by activated AMPK. Metformin, AICAR or berberine all were shown to diminish neutrophil and macrophage proinflammatory activation, as well as to decrease the severity of endotoxin- or ventilator-induced acute lung injury (29,31,58). Exposure of macrophages to antiinflammatory mediators, such as IL-10 or TGF-β, resulted in activation of AMPK followed by transition of the cells from the M1 to M2 phenotype (33). Recent studies have shown that activated AMPK can also modulate the resolution of inflammatory conditions due to enhancement of the phagocytic ability of macrophages and neutrophils. In particular, AMPK activation increased the uptake of bacteria and enhanced efferocytosis, an essential process in the resolution of inflammation in which apoptotic cells are ingested and cleared by phagocytic cells (39,59,60). Beneficial effects of AMPK activation also were related to improvement of vascular integrity in mice models for endotoxemia-induced ALI and to airway remodeling in asthma (34,61). In experimental models of diabetes, endothelial barrier function was preserved through mechanisms involving activation of AMPK (62).
Recent studies have shown that AMPK phosphorylation was diminished upon exposure of cells to LPS. In particular, culture with LPS significantly decreased AMPK activity in neutrophils, peritoneal macrophages, Raw 264.7 cells, and endothelial cells (33,34,45,63,64). The combination of LPS and saturated fatty acid palmitate also was shown to induce prolonged inactivation of AMPK in bone marrow macrophages (65). Our present results indicate that in spite of inhibitory action of LPS, inclusion of AICAR or metformin was able to partially increase AMPK phosphorylation, even when included in cultures after cellular exposure to LPS (Figure 3) (45).
Bacterial dissemination leading to multiorgan injury contributes to the high mortality rate associated with sepsis (10,11,19). Whereas many patients survive the initial stages of sepsis, many will develop later clinical complications including nosocomial infections that lead to prolonged hospitalization. In spite of improved understanding of the mechanisms responsible for complications in sepsis, pharmacological approaches to prevent secondary infection and decrease morbidity and mortality associated with late infection have not been well characterized (57). Metformin is approved for use in patients with diabetes with a well-established safety profile and known side effects and, for these reasons, may be considered for examination as a therapeutic approach in clinical trials of patients with sepsis. Although the early use of antibiotics is beneficial during sepsis, microbial products, as well as hostderived danger associated molecular pattern molecules (DAMPs) are still frequently present in the circulation and are likely to contribute to organ system dysfunction (66,67). For example, release of HMGB1, histones and mitochondrial proteins are known to increase the severity of acute inflammatory conditions and intensify the immunosuppressed state characteristic of late sepsis (66, 67, 68, 69, 70, 71). Of note, AMPK activators, including metformin, were shown to diminish acute inflammatory injury of lung or liver as well as decrease the release of DAMPs and improve mortality in experimental models of LPS-induced sepsis (5,29,36). Although antibiotics are sufficient to kill bacteria, the combination of metformin and antibiotics may have additional benefit in sepsis, particularly as metforminstimulated neutrophils have increased chemotaxis and bacterial uptake.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported in part by National Institutes of Health Grants GM87748 and HL107585 to JW Zmijewski and NIH P30 AR 48311 to The Flow Cy-tometry Core, Arthritis and Muscu-loskeletal Center UAB. We thank Anna A Zmijewska and Enid Keyser for technical support.
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