Fibrosis is a pathological result of a dysfunctional repair response to tissue injury and occurs in a number of organs, including the lungs. Cellular metabolism regulates tissue repair and remodelling responses to injury. AMPK is a critical sensor of cellular bioenergetics and controls the switch from anabolic to catabolic metabolism. However, the role of AMPK in fibrosis is not well understood. Here, we demonstrate that in humans with idiopathic pulmonary fibrosis (IPF) and in an experimental mouse model of lung fibrosis, AMPK activity is lower in fibrotic regions associated with metabolically active and apoptosis-resistant myofibroblasts. Pharmacological activation of AMPK in myofibroblasts from lungs of humans with IPF display lower fibrotic activity, along with enhanced mitochondrial biogenesis and normalization of sensitivity to apoptosis. In a bleomycin model of lung fibrosis in mice, metformin therapeutically accelerates the resolution of well-established fibrosis in an AMPK-dependent manner. These studies implicate deficient AMPK activation in non-resolving, pathologic fibrotic processes, and support a role for metformin (or other AMPK activators) to reverse established fibrosis by facilitating deactivation and apoptosis of myofibroblasts.

Although neutrophil extracellular traps (NETs) form to prevent dissemination of pathogenic microorganisms, excessive release of DNA and DNA-associated proteins can also perpetuate sterile inflammation. In this study, we found that the danger-associated molecular pattern protein high-mobility group box 1 (HMGB1) can induce NET formation. NET formation was found after exposure of wild-type and receptor for advanced glycation end products-deficient neutrophil to HMGB1, whereas deficiency of Toll-like receptor (TLR)4 diminished the ability of neutrophils to produce NETs. Incubation of neutrophils with HMGB1 significantly increased the amount of DNA and histone 3 released as well as intracellular histone 3 citrullination, a signaling event that precedes chromatin decondensation. In vivo, neutrophils isolated from bronchoalveolar lavages of mice exposed to LPS and HMGB1 showed consistently greater ability to produce NETs compared with pulmonary neutrophils from mice that received LPS alone. In contrast, mice treated with LPS and neutralizing antibody to HMGB1 had decreased amounts of the inflammatory cytokines TNF-α and macrophage inflammatory protein 2, as well as of free DNA and histone 3 in bronchoalveolar lavage fluids. Airway neutrophils from LPS-exposed mice that had been treated with anti-HMGB1 antibodies showed decreased citrullination of histone 3. These results demonstrate that interactions between HMGB1 and TLR4 enhance the formation of NETs and provide a novel mechanism through which HMGB1 may contribute to the severity of neutrophil-associated inflammatory conditions.

Although resistin was recently found to modulate insulin resistance in preclinical models of type II diabetes and obesity, recent studies have also suggested that resistin has proinflammatory properties. In these studies, we examined if the human specific variant of resistin affects neutrophil activation as well as the severity of LPS-induced acute lung injury (ALI). Because human and mouse resistin have distinct patterns of tissue distribution, experiments were performed using resistin humanized mice that exclusively express human resistin (hRTN+/−/−), but are deficient in mouse resistin. Enhanced production of TNF-α or MIP-2 was found in LPS-treated hRtn+/−/−, compared to control Rtn−/−/− neutrophils. Expression of human resistin inhibited the activation of AMP-activated protein kinase (AMPK), a major sensor and regulator of cellular bioenergetics that is also implicated in inhibiting inflammatory activity of neutrophils and macrophages. In addition to the ability of resistin to sensitize neutrophils to LPS stimulation, human resistin also enhanced neutrophil extracellular trap formation. In LPS-induced ALI, humanized resistin mice demonstrated enhanced production of pro-inflammatory cytokines, more severe pulmonary edema, increased NET formation, and elevated concentration of the alarmins HMGB1 and histone 3 in the lungs. Our results suggest that human resistin may play an important contributory role in enhancing TLR4 induced inflammatory responses, and may be a target for future therapies aimed at diminishing the severity of acute lung injury and other inflammatory situations where neutrophils play a major role.

Although AMP-activated protein kinase (AMPK) is involved in regulating carbohydrate and lipid metabolism, activated AMPK also plays an anti-inflammatory role in many cell populations. However, despite the ability of AMPK activation to diminish the severity of inflammatory responses, previous studies have found that AMPK activity is diminished in LPS-treated neutrophils and also in lungs of mice with LPS-induced acute lung injury (ALI). Since GSK3β participates in regulating AMPK activity, we examined potential roles for GSK3β in modulating LPS-induced activation of neutrophils and macrophages and in influencing severity of ALI. We found that GSK3β-dependent phosphorylation of T479-AMPK was associated with pT172 dephosphorylation and inactivation of AMPK following TLR4 engagement. GSK3β inhibitors BIO (6-bromoindirubin-3'-oxime), SB216763, or siRNA knockdown of GSK3β, but not the PI3K/AKT inhibitor LY294002, prevented Thr172-AMPK dephosphorylation. Exposure to LPS resulted in rapid binding between IKKβ and AMPKα, and phosphorylation of S485-AMPK by IKKβ. These results suggest that IKKβ-dependent phosphorylation of S485-AMPK was an essential step in subsequent phosphorylation and inactivation AMPK by GSK3β. Inhibition of GSK3β activity delayed IκBα degradation and diminished expression of the proinflammatory TNF-α in LPS-stimulated neutrophils and macrophages. In vivo, inhibition of GSK3β decreased the severity of LPS-induced lung injury as assessed by development of pulmonary edema, production of TNF-α and MIP-2, and release of the alarmins HMGB1 and histone 3 in the lungs. These results show that inhibition of AMPK by GSK3β plays an important contributory role in enhancing LPS-induced inflammatory responses, including worsening the severity of ALI.

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

Alterations in metabolic and bioenergetic homeostasis contribute to sepsis-mediated organ injury. However, how AMP-activated protein kinase (AMPK), a major sensor and regulator of energy expenditure and production, affects development of organ injury and loss of innate capacity during polymicrobial sepsis remains unclear. In the present experiments, we found that cross-talk between the AMPK and GSK3β signaling pathways controls chemotaxis and the ability of neutrophils and macrophages to kill bacteria ex vivo. In mice with polymicrobial abdominal sepsis or more severe sepsis induced by the combination of hemorrhage and intraabdominal infection, administration of the AMPK activator metformin or the GSK3β inhibitor SB216763 reduced the severity of acute lung injury (ALI). Improved survival in metformin-treated septic mice was correlated with preservation of mitochondrial complex V (ATP synthase) function and increased amounts of ETC complex III and IV. Although immunosuppression is a consequence of sepsis, metformin effectively increased innate immune capacity to eradicate P. aeruginosa in the lungs of septic mice. We also found that AMPK activation diminished accumulation of the immunosuppressive transcriptional factor HIF-1α as well as the development of endotoxin tolerance in LPS-treated macrophages. Furthermore, AMPK-dependent preservation of mitochondrial membrane potential also prevented LPS-mediated dysfunction of neutrophil chemotaxis. These results indicate that AMPK activation reduces the severity of polymicrobial sepsis-induced lung injury and prevents the development of sepsis-associated immunosuppression.

Background: Billions of cells undergo apoptosis in the human body every day, and the removal of dying cells (efferocytosis) is essential for tissue homeostasis. Results: Energy demand during efferocytosis results in AMPK activation followed by enhancement of macrophage chemokinesis and efferocytosis. Conclusion: The AMPK pathway is intimately linked to cellular bioenergetics and efferocytosis. Significance: A novel mechanism of mitochondria and AMPK-dependent stimulation of efferocytosis is proposed.

We aimed to identify miRNAs whose expression levels in fetal tissues are altered by exposure to a diabetic milieu and elucidate the impact on target protein expression. Gestational diabetes mellitus (GDM) affects both immediate and future disease risk in the offspring. We hypothesized that GDM alters miRNA expression in human umbilical vein endothelial cells (HUVECs) that may influence metabolic processes. A cross-sectional design compared differences in miRNA expression in HUVECs and target protein abundance in placentae between infants of women with GDM (IGDM) and infants born to normoglycaemic controls. miRNAs were identified using microarray profiling and literature review and validated by quantitative PCR (qPCR). In vitro transfection studies explored the impact of the miRNA on target protein expression. Expression of seven miRNA species, miR-30c-5p, miR-452-5p, miR-126-3p, miR-130b-3p, miR-148a-3p, miR-let-7a-5p and miR-let-7g-5p, was higher in the HUVECs of IGDM. Abundance of the catalytic subunit of AMP-activated protein kinase α1 (AMPKα1) was decreased in the HUVECs and BeWo cells (transformed trophoblast cell line) transfected with miR-130b and miR-148a mimics. AMPKα1 expression was also decreased in placental tissues of IGDM. The expression of several miRNAs were altered by in utero exposure to DM in infants of women whose dysglycaemia was very well controlled by current standards. Decreased expression of AMPKα1 as a result of increased levels of miR-130b and miR-148a may potentially explain the decrease in fat oxidation we reported in infants at 1 month of age and, if persistent, may predispose offspring to future metabolic disease.