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.

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.

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.