Metabolic and Bioenergetic Alterations are Associated with Infection Susceptibility in Survivors of Severe Trauma: An Exploratory Study.

BACKGROUND: Trauma and blood loss are frequently associated with organ failure, immune dysfunction, and a high risk of secondary bacterial lung infections. We aim to test if plasma metabolomic flux and monocyte bioenergetics are altered in association with trauma and related secondary infections. METHODS: Plasma samples were collected from trauma patients at three time points: days 0, 3, and 7 post-admission. Metabolites (140) were measured in plasma from trauma survivors (n = 24) and healthy control individuals (HC, n = 10). Further analysis within the trauma cohort included subsets of trauma/infection-negative (TIneg, n = 12) and trauma/infection-positive patients (TIpos, n = 12). The bioenergetic profile in monocytes was determined using mitochondrial and glycolytic stress tests. RESULTS: In the trauma cohort, significant alterations were observed in 29 metabolites directly affecting 11 major metabolic pathways, while 34 metabolite alterations affected 8 pathways in TIpos, versus TIneg patients. The most altered metabolic pathways included protein synthesis, the urea cycle/arginine metabolism, phenylalanine, tyrosine, tryptophan biosynthesis, and carnitine compound family. In monocytes from trauma patients, reduced mitochondrial indices and loss of glycolytic plasticity were consistent with an altered profile of plasma metabolites in the TCA cycle and glycolysis. CONCLUSIONS: Our study highlights that the metabolic profile is significantly and persistently affected by trauma and related infections. Among trauma survivors, metabolic alterations in plasma were associated with reduced monocyte bioenergetics. These exploratory findings establish a groundwork for future clinical studies aimed at enhancing our understanding of the interplay between metabolic/bioenergetic alterations associated with trauma and secondary bacterial infections.

PAI-1 Regulation of p53 Expression and Senescence in Type II Alveolar Epithelial Cells.

Cellular senescence contributes importantly to aging and aging-related diseases, including idiopathic pulmonary fibrosis (IPF). Alveolar epithelial type II (ATII) cells are progenitors of alveolar epithelium, and ATII cell senescence is evident in IPF. Previous studies from this lab have shown that increased expression of plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor, promotes ATII cell senescence through inducing p53, a master cell cycle repressor, and activating p53-p21-pRb cell cycle repression pathway. In this study, we further show that PAI-1 binds to proteasome components and inhibits proteasome activity and p53 degradation in human lung epithelial A549 cells and primary mouse ATII cells. This is associated with a senescence phenotype of these cells, manifested as increased p53 and p21 expression, decreased phosphorylated retinoblastoma protein (pRb), and increased senescence-associated beta-galactose (SA-ß-gal) activity. Moreover, we find that, although overexpression of wild-type PAI-1 (wtPAI-1) or a secretion-deficient, mature form of PAI-1 (sdPAI-1) alone induces ATII cell senescence (increases SA-ß-gal activity), only wtPAI-1 induces p53, suggesting that the premature form of PAI-1 is required for the interaction with the proteasome. In summary, our data indicate that PAI-1 can bind to proteasome components and thus inhibit proteasome activity and p53 degradation in ATII cells. As p53 is a master cell cycle repressor and PAI-1 expression is increased in many senescent cells, the results from this study will have a significant impact not only on ATII cell senescence/lung fibrosis but also on the senescence of other types of cells in different diseases.

AMPK activation improves recovery from pneumonia-induced lung injury via reduction of er-stress and apoptosis in alveolar epithelial cells.

BACKGROUND: Bacterial pneumonia and related lung injury are among the most frequent causes of mortality in intensive care units, but also inflict serious and prolonged respiratory complications among survivors. Given that endoplasmic reticulum (ER) stress is a hallmark of sepsis-related alveolar epithelial cell (AEC) dysfunction, we tested if AMP-activated protein kinase (AMPK) affects recovery from ER stress and apoptosis of AECs during post-bacterial infection. METHODS: In a murine model of lung injury by P. aeruginosa non-lethal infection, therapeutic interventions included AMPK activator metformin or GSK-3ß inhibitor Tideglusib for 96 h. Recovery from AEC injury was evidenced by accumulation of soluble T-1α (AEC Type 1 marker) in BAL fluids along with fluorescence analysis of ER-stress (CHOP) and apoptosis (TUNEL) in lung sections. AMPK phosphorylation status and mediators of ER stress were determined via Immunoblot analysis from lung homogenates. Macrophage-dependent clearance of apoptotic cells was determined using flow cytometry assay. RESULTS: P. aeruginosa-induced lung injury resulted in accumulation of neutrophils and cellular debris in the alveolar space along with persistent (96 h) ER-stress and apoptosis of AECs. While lung infection triggered AMPK inactivation (de-phosphorylation of Thr172-AMPK), metformin and Tideglusib promptly restored the AMPK activation status. In post infected mice, AMPK activation reduced indices of lung injury, ER stress and related apoptosis of AECs, as early as 24 h post administration of AMPK activators. In addition, we demonstrate that the extent of apoptotic cell accumulation is also dependent on AMPK-mediated clearance of apoptotic cells by macrophages. CONCLUSIONS: Our study provides important insights into AMPK function in the preservation of AEC viability after bacterial infection, in particular due reduction of ER-stress and apoptosis, thereby promoting effective recovery from lung injury after pneumonia.

Mitochondrial uncoupling protein-2 reprograms metabolism to induce oxidative stress and myofibroblast senescence in age-associated lung fibrosis.

Mitochondrial dysfunction has been associated with age-related diseases, including idiopathic pulmonary fibrosis (IPF). We provide evidence that implicates chronic elevation of the mitochondrial anion carrier protein, uncoupling protein-2 (UCP2), in increased generation of reactive oxygen species, altered redox state and cellular bioenergetics, impaired fatty acid oxidation, and induction of myofibroblast senescence. This pro-oxidant senescence reprogramming occurs in concert with conventional actions of UCP2 as an uncoupler of oxidative phosphorylation with dissipation of the mitochondrial membrane potential. UCP2 is highly expressed in human IPF lung myofibroblasts and in aged fibroblasts. In an aging murine model of lung fibrosis, the in vivo silencing of UCP2 induces fibrosis regression. These studies indicate a pro-fibrotic function of UCP2 in chronic lung disease and support its therapeutic targeting in age-related diseases associated with impaired tissue regeneration and organ fibrosis.

ZKSCAN3 in severe bacterial lung infection and sepsis-induced immunosuppression.

The mortality rates among patients who initially survive sepsis are, in part, associated with a high risk of secondary lung infections and respiratory failure. Given that phagolysosomes are important for intracellular killing of pathogenic microbes, we investigated how severe lung infections associated with post-sepsis immunosuppression affect phagolysosome biogenesis. In mice with P. aeruginosa-induced pneumonia, we found a depletion of both phagosomes and lysosomes, as evidenced by decreased amounts of microtubule associated protein light chain 3-II (LC3-II) and lysosomal-associated membrane protein (LAMP1). We also found a loss of transcription factor E3 (TFE3) and transcription factor EB (TFEB), which are important activators for transcription of genes encoding autophagy and lysosomal proteins. These events were associated with increased expression of ZKSCAN3, a repressor for transcription of genes encoding autophagy and lysosomal proteins. Zkscan3-/- mice had increased expression of genes involved in the autophagy-lysosomal pathway along with enhanced killing of P. aeruginosa in the lungs, as compared to wild-type mice. These findings highlight the involvement of ZKSCAN3 in response to severe lung infection, including susceptibility to secondary bacterial infections due to immunosuppression.

Mesenchymal stromal cell aging impairs the self-organizing capacity of lung alveolar epithelial stem cells.

Multicellular organisms maintain structure and function of tissues/organs through emergent, self-organizing behavior. In this report, we demonstrate a critical role for lung mesenchymal stromal cell (L-MSC) aging in determining the capacity to form three-dimensional organoids or 'alveolospheres' with type 2 alveolar epithelial cells (AEC2s). In contrast to L-MSCs from aged mice, young L-MSCs support the efficient formation of alveolospheres when co-cultured with young or aged AEC2s. Aged L-MSCs demonstrated features of cellular senescence, altered bioenergetics, and a senescence-associated secretory profile (SASP). The reactive oxygen species generating enzyme, NADPH oxidase 4 (Nox4), was highly activated in aged L-MSCs and Nox4 downregulation was sufficient to, at least partially, reverse this age-related energy deficit, while restoring the self-organizing capacity of alveolospheres. Together, these data indicate a critical role for cellular bioenergetics and redox homeostasis in an organoid model of self-organization and support the concept of thermodynamic entropy in aging biology.

Many tissues in the body are capable of regenerating by replacing defective or worn-out cells with new ones. This process relies heavily on stem cells, which are precursor cells that lack a set role in the body and can develop into different types of cells under the right conditions. Tissues often have their own pool of stem cells that they use to replenish damaged cells. But as we age, this regeneration process becomes less effective. Many of our organs, such as the lungs, are lined with epithelial cells. These cells form a protective barrier, controlling what substances get in and out of the tissue. Alveoli are parts of the lungs that allow oxygen and carbon dioxide to move between the blood and the air in the lungs. And alveoli rely on an effective epithelial cell lining to work properly. To replenish these epithelial cells, alveoli have pockets, in which a type of epithelial cell, known as AEC2, lives. These cells can serve as stem cells, developing into a different type of cell under the right conditions. To work properly, AEC2 cells require close interactions with another type of cell called L-MSC, which supports the maintenance of other cells and also has the ability to differentiate into several other cell types. Both cell types can be found close together in these stem cell pockets. So far, it has been unclear how aging affects how these cells work together to replenish the epithelial lining of the alveoli. To investigate, Chanda et al. probed AEC2s and L-MSCs in the alveoli of young and old mice. The researchers collected both cell types from young (2-3 months) and aged (22-24 months) mice. Various combinations of these cells were grown to form 3D structures, mimicking how the cells grow in the lungs. Young L-MSCs formed normal 3D structures with both young and aged AEC2 cells. But aged L-MSCs developed abnormal, loose structures with AEC2 cells (both young and old cells). Aged L-MSCs were found to have higher levels of an enzyme (called Nox4) that produces oxidants and other 'pro-aging' factors, compared to young L-MSCs. However, reducing Nox4 levels in aged L-MSCs allowed these cells to form normal 3D structures with young AEC2 cells, but not aged AEC2 cells. These findings highlight the varying effects specific stem cells have, and how their behaviour is affected by pro-aging factors. Moreover, the pro-aging enzyme Nox4 shows potential as a therapeutic target ­ downregulating its activity may reverse critical effects of aging in cells.

Restoration of SIRT3 gene expression by airway delivery resolves age-associated persistent lung fibrosis in mice.

Aging is a risk factor for progressive fibrotic disorders involving diverse organ systems, including the lung. Idiopathic pulmonary fibrosis, an age-associated degenerative lung disorder, is characterized by persistence of apoptosis-resistant myofibroblasts. In this report, we demonstrate that sirtuin-3 (SIRT3), a mitochondrial deacetylase, is downregulated in lungs of IPF human subjects and in mice subjected to lung injury. Over-expression of the SIRT3 cDNA via airway delivery restored capacity for fibrosis resolution in aged mice, in association with activation of the forkhead box transcription factor, FoxO3a, in fibroblasts, upregulation of pro-apoptotic members of the Bcl-2 family, and recovery of apoptosis susceptibility. While transforming growth factor-ß1 reduced levels of SIRT3 and FoxO3a in lung fibroblasts, cell non-autonomous effects involving macrophage secreted products were necessary for SIRT3-mediated activation of FoxO3a. Together, these findings reveal a novel role of SIRT3 in pro-resolution macrophage functions that restore susceptibility to apoptosis in fibroblasts via a FoxO3a-dependent mechanism.

AMPK activates Parkin independent autophagy and improves post sepsis immune defense against secondary bacterial lung infections.

Metabolic and bioenergetic plasticity of immune cells is essential for optimal responses to bacterial infections. AMPK and Parkin ubiquitin ligase are known to regulate mitochondrial quality control mitophagy that prevents unwanted inflammatory responses. However, it is not known if this evolutionarily conserved mechanism has been coopted by the host immune defense to eradicate bacterial pathogens and influence post-sepsis immunosuppression. Parkin, AMPK levels, and the effects of AMPK activators were investigated in human leukocytes from sepsis survivors as well as wild type and Park2-/- murine macrophages. In vivo, the impact of AMPK and Parkin was determined in mice subjected to polymicrobial intra-abdominal sepsis and secondary lung bacterial infections. Mice were treated with metformin during established immunosuppression. We showed that bacteria and mitochondria share mechanisms of autophagic killing/clearance triggered by sentinel events that involve depolarization of mitochondria and recruitment of Parkin in macrophages. Parkin-deficient mice/macrophages fail to form phagolysosomes and kill bacteria. This impairment of host defense is seen in the context of sepsis-induced immunosuppression with decreased levels of Parkin. AMPK activators, including metformin, stimulate Parkin-independent autophagy and bacterial killing in leukocytes from post-shock patients and in lungs of sepsis-immunosuppressed mice. Our results support a dual role of Parkin and AMPK in the clearance of dysfunctional mitochondria and killing of pathogenic bacteria, and explain the immunosuppressive phenotype associated Parkin and AMPK deficiency. AMPK activation appeared to be a crucial therapeutic target for the macrophage immunosuppressive phenotype and to reduce severity of secondary bacterial lung infections and respiratory failure.

NETosis in the pathogenesis of acute lung injury following cutaneous chemical burns.

Despite the high morbidity and mortality among patients with extensive cutaneous burns in the intensive care unit due to the development of acute respiratory distress syndrome, effective therapeutics remain to be determined. This is primarily because the mechanisms leading to acute lung injury (ALI) in these patients remain unknown. We test the hypothesis that cutaneous chemical burns promote lung injury due to systemic activation of neutrophils, in particular, toxicity mediated by the deployment of neutrophil extracellular traps (NETs). We also demonstrate the potential benefit of a peptidyl arginine deiminase 4 (PAD4) inhibitor to prevent NETosis and to preserve microvascular endothelial barrier function, thus reducing the severity of ALI in mice. Our data demonstrated that phenylarsine oxide (PAO) treatment of neutrophils caused increased intracellular Ca2+-associated PAD4 activity. A dermal chemical burn by lewisite or PAO resulted in PAD4 activation, NETosis, and ALI. NETs disrupted the barrier function of endothelial cells in human lung microvascular endothelial cell spheroids. Citrullinated histone 3 alone caused ALI in mice. Pharmacologic or genetic abrogation of PAD4 inhibited lung injury following cutaneous chemical burns. Cutaneous burns by lewisite and PAO caused ALI by PAD4-mediated NETosis. PAD4 inhibitors may have potential as countermeasures to suppress detrimental lung injury after chemical burns.

Bioenergetic maladaptation and release of HMGB1 in calcineurin inhibitor-mediated nephrotoxicity.

Calcineurin inhibitors (CNIs) are potent immunosuppressive agents, universally used following solid organ transplantation to prevent rejection. Although effective, the long-term use of CNIs is associated with nephrotoxicity. The etiology of this adverse effect is complex, and effective therapeutic interventions remain to be determined. Using a combination of in vitro techniques and a mouse model of CNI-mediated nephrotoxicity, we found that the CNIs, cyclosporine A (CsA), and tacrolimus (TAC) share a similar mechanism of tubular epithelial kidney cell injury, including mitochondrial dysfunction and release of High-Mobility Group Box I (HMGB1). CNIs promote bioenergetic reprogramming due to mitochondrial dysfunction and a shift toward glycolytic metabolism. These events were accompanied by diminished cell-to-cell adhesion, loss of the epithelial cell phenotype, and release of HMGB1. Notably, Erk1/2 inhibitors effectively diminished HMGB1 release, and similar inhibitor was observed on inclusion of pan-caspase inhibitor zVAD-FMK. In vivo, while CNIs activate tissue proremodeling signaling pathways, MAPK/Erk1/2 inhibitor prevented nephrotoxicity, including diminished HMGB1 release from kidney epithelial cells and accumulation in urine. In summary, HMGB1 is an early indicator and marker of progressive nephrotoxicity induced by CNIs. We suggest that proremodeling signaling pathway and loss of mitochondrial redox/bioenergetics homeostasis are crucial therapeutic targets to ameliorate CNI-mediated nephrotoxicity.

Protective role of HO-1 against acute kidney injury caused by cutaneous exposure to arsenicals.

Lewisite and many other similar arsenicals are warfare vesicants developed and weaponized for use in World Wars I and II. These chemicals, when exposed to the skin and other epithelial tissues, cause rapid severe inflammation and systemic damage. Here, we show that topically applied arsenicals in a murine model produce significant acute kidney injury (AKI), as determined by an increase in the AKI biomarkers NGAL and KIM-1. An increase in reactive oxygen species and ER stress proteins, such as ATF4 and CHOP, correlated with the induction of these AKI biomarkers. Also, TUNEL staining of CHOP-positive renal tubular cells suggests CHOP mediates apoptosis in these cells. A systemic inflammatory response characterized by a significant elevation in inflammatory mediators, such as IL-6, IFN-α, and COX-2, in the kidney could be the underlying cause of AKI. The mechanism of arsenical-mediated inflammation involves activation of AMPK/Nrf2 signaling pathways, which regulate heme oxygenase-1 (HO-1). Indeed, HO-1 induction with cobalt protoporphyrin (CoPP) treatment in arsenical-treated HEK293 cells afforded cytoprotection by attenuating CHOP-associated apoptosis and cytokine mRNA levels. These results demonstrate that topical exposure to arsenicals causes AKI and that HO-1 activation may serve a protective role in this setting.

Oxidative cross-linking of fibronectin confers protease resistance and inhibits cellular migration.

The oxidation of tyrosine residues to generate o,o'-dityrosine cross-links in extracellular proteins is necessary for the proper function of the extracellular matrix (ECM) in various contexts in invertebrates. Tyrosine oxidation is also required for the biosynthesis of thyroid hormone in vertebrates, and there is evidence for oxidative cross-linking reactions occurring in extracellular proteins secreted by myofibroblasts. The ECM protein fibronectin circulates in the blood as a globular protein that dimerizes through disulfide bridges generated by cysteine oxidation. We found that cellular (fibrillar) fibronectin on the surface of transforming growth factor-ß1 (TGF-ß1)-activated human myofibroblasts underwent multimerization by o,o'-dityrosine cross-linking under reducing conditions that disrupt disulfide bridges, but soluble fibronectin did not. This reaction on tyrosine residues required both the TGF-ß1-dependent production of hydrogen peroxide and the presence of myeloperoxidase (MPO) derived from inflammatory cells, which are active participants in wound healing and fibrogenic processes. Oxidative cross-linking of matrix fibronectin attenuated both epithelial and fibroblast migration and conferred resistance to proteolysis by multiple proteases. The abundance of circulating o,o'-dityrosine-modified fibronectin was increased in a murine model of lung fibrosis and in human subjects with interstitial lung disease compared to that in control healthy subjects. These studies indicate that tyrosine can undergo stable, covalent linkages in fibrillar fibronectin under inflammatory conditions and that this modification affects the migratory behavior of cells on such modified matrices, suggesting that this modification may play a role in both physiologic and pathophysiologic tissue repair.

NOX2 decoy peptides disrupt trauma-mediated neutrophil immunosuppression and protect against lethal peritonitis.

Trauma and sepsis are frequent causes of immunosuppression and risk of secondary bacterial infections and mortality among critically ill patients. Reduced activity of neutrophil NADPH oxidase 2 (NOX2) and impaired bacterial killing are among the major indices of immunosuppression. We hypothesize that NOX2-decoy peptides disrupt the inhibition of neutrophil NOX2 by plasma of patients with severe trauma and immunosuppression, thereby preserving the neutrophil respiratory burst that is a central antimicrobial mechanism. We demonstrate that plasma from trauma/hemorrhage (T/H) patients, but not healthy donors (HD), significantly reduced the activity of neutrophil NOX2 and impaired bacterial killing. The inhibitory action of plasma was associated with an increase in bacterial infections among trauma survivors. High Mobility Group Box 1 (HMGB1) is a mediator of lethality in trauma and sepsis and our mechanistic studies revealed that disulfide and oxidized forms of HMGB1 bind to the gp91phox subunit of NOX2, and thus decrease the neutrophil respiratory burst and bacterial killing. NOX2 decoy Anti-Immunosuppression (Ai) Peptides 1 and 3 effectively disrupted the immunosuppressive action of T/H plasma. HMGB1 selectively binds to Ai-Peptide 3, supporting the possibility for direct interaction between HMGB1 and the third external loop of gp91phox. In vivo, Ai-Peptides improved survival of mice subjected to lethal peritonitis. Taken together, plasma-dependent inhibition of neutrophil NOX2 appeared to be a suitable indicator of immunosuppression in patients with severe trauma. Given that gp91phox decoys protected the neutrophil respiratory burst, selected Ai-Peptides have therapeutic potential to reduce bacterial infections and end-organ injury associated with sepsis/trauma-induced immunosuppression.

SIRT3 diminishes inflammation and mitigates endotoxin-induced acute lung injury.

Acute lung injury (ALI) is characterized by exuberant proinflammatory responses and mitochondrial dysfunction. However, the link between mitochondrial dysfunction and inflammation in ALI is not well understood. In this report, we demonstrate a critical role for the mitochondrial NAD+-dependent deacetylase, sirtuin-3 (SIRT3), in regulating macrophage mitochondrial bioenergetics, ROS formation, and proinflammatory responses. We found that SIRT3 expression was significantly diminished in lungs of mice subjected to LPS-induced ALI. SIRT3-deficient mice (SIRT3-/-) develop more severe ALI compared with wild-type controls (SIRT3+/+). Macrophages obtained from SIRT3-/- mice show significant alterations in mitochondrial bioenergetic and redox homeostasis, in association with a proinflammatory phenotype characterized by NLRP3 inflammasome activation. The SIRT3 activator viniferin restored macrophage bioenergetic function in LPS-treated macrophages. Viniferin also reduced NLRP3 activation and the production of proinflammatory cytokines, effects that were absent in SIRT3-/- macrophages. In-vivo administration of viniferin reduced production of inflammatory mediators TNF-α, MIP-2, IL-6, IL-1ß, and HMGB1, and diminished neutrophil influx and severity of endotoxin-mediated ALI; this protective effect of vinferin was abolished in SIRT3-/- mice. Taken together, our results show that the induction/activation of SIRT3 may serve as a new therapeutic strategy in ALI by modulating cellular bioenergetics, controlling inflammatory responses, and reducing the severity of lung injury.

Author Correction: Metformin reverses established lung fibrosis in a bleomycin model.

In the version of this article originally published, a grant was omitted from the Acknowledgements section. The following sentence should have been included: "R.B.M. was supported by a Department of Veterans Affairs Merit Award (5I01BX003272)." The error has been corrected in the HTML and PDF versions of this article.

Metformin reverses established lung fibrosis in a bleomycin model.

Fibrosis is a pathological result of a dysfunctional repair response to tissue injury and occurs in a number of organs, including the lungs1. Cellular metabolism regulates tissue repair and remodelling responses to injury2-4. AMPK is a critical sensor of cellular bioenergetics and controls the switch from anabolic to catabolic metabolism5. 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.

Impaired efferocytosis and neutrophil extracellular trap clearance by macrophages in ARDS.

Exaggerated release of neutrophil extracellular traps (NETs) along with decreased NET clearance and inability to remove apoptotic cells (efferocytosis) may contribute to sustained inflammation in acute respiratory distress syndrome (ARDS). Recent studies in experimental models of ARDS have revealed the crosstalk between AMP-activated protein kinase (AMPK) and high-mobility group box 1 (HMGB1), which may contribute to effectiveness of efferocytosis, thereby reducing inflammation and ARDS severity.We investigated neutrophil and NET clearance by macrophages from control and ARDS patients and examined how bronchoalveolar lavage (BAL) fluid from control and ARDS patients could affect NET formation and efferocytosis. Metformin (an AMPK activator) and neutralising antibody against HMGB1 were applied to improve efferocytosis and NET clearance.Neutrophils from ARDS patients showed significantly reduced apoptosis. Conversely, NET formation was significantly enhanced in ARDS patients. Exposure of neutrophils to ARDS BAL fluid promoted NET production, while control BAL fluid had no effect. Macrophage engulfment of NETs and apoptotic neutrophils was diminished in ARDS patients. Notably, activation of AMPK in macrophages or neutralisation of HMGB1 in BAL fluid improved efferocytosis and NET clearance.In conclusion, restoration of AMPK activity with metformin or specific neutralisation of HMGB1 in BAL fluid represent promising therapeutic strategies to decrease sustained lung inflammation during ARDS.