Visiting Instructor/Lecturer, Pediatrics - Critical Care
Cell-Free Hemoglobin in Acute Kidney Injury after Lung Transplantation and Experimental Renal Ischemia/Reperfusion.
International journal of molecular sciences
2022; 23 (21)
Cell-free hemoglobin (CFH), a pro-oxidant and cytotoxic compound that is released in hemolysis, has been associated with nephrotoxicity. Lung transplantation (LuTx) is a clinical condition with a high incidence of acute kidney injury (AKI). In this study, we investigated the plasma levels of CFH and haptoglobin, a CFH-binding serum protein, in prospectively enrolled LuTx patients (n = 20) with and without AKI. LuTx patients with postoperative AKI had higher CFH plasma levels at the end of surgery compared with no-AKI patients, and CFH correlated with serum creatinine at 48 h. Moreover, CFH levels inversely correlated with haptoglobin levels, which were significantly reduced at the end of surgery in LuTx patients with AKI. Because multiple other factors can contribute to AKI development in the complex clinical setting of LuTx, we next investigated the role of exogenous CFH administration in a mouse model of mild bilateral renal ischemia reperfusion injury (IRI). Exogenous administration of CFH after reperfusion caused overt AKI with creatinine increase, tubular injury, and enhanced markers of renal inflammation compared with vehicle-treated animals. In conclusion, CFH is a possible factor contributing to postoperative AKI after LuTx and promotes AKI in an experimental model of mild transient renal ischemia. Targeting CFH might be a therapeutic option to prevent AKI after LuTx.
View details for DOI 10.3390/ijms232113272
View details for PubMedID 36362059
Genetic BACH1 deficiency alters mitochondrial function and increases NLRP3 inflammasome activation in mouse macrophages.
2022; 51: 102265
BTB-and-CNC homologue 1 (BACH1), a heme-regulated transcription factor, mediates innate immune responses via its functional role in macrophages. BACH1 has recently been shown to modulate mitochondrial metabolism in cancer cells. In the current study, we utilized a proteomics approach and demonstrate that genetic deletion of BACH1 in mouse macrophages is associated with decreased levels of various mitochondrial proteins, particularly mitochondrial complex I. Bioenergetic studies revealed alterations of mitochondrial energy metabolism in BACH1-/- macrophages with a shift towards increased glycolysis and decreased oxidative phosphorylation. Moreover, these cells exhibited enhanced mitochondrial membrane potential and generation of mitochondrial reactive oxygen species (mtROS) along with lower levels of mitophagy. Notably, a higher inducibility of NLRP3 inflammasome activation in response to ATP and nigericin following challenge with lipopolysaccharide (LPS) was observed in BACH1-deficient macrophages compared to wild-type cells. Mechanistically, pharmacological inhibition of mtROS markedly attenuated inflammasome activation. In addition, it is shown that inducible nitric oxide synthase and cyclooxygenase-2, both of which are markedly induced by LPS in macrophages, are directly implicated in BACH1-dependent regulation of NLRP3 inflammasome activation. Taken together, the current findings indicate that BACH1 is critical for immunomodulation of macrophages and may serve as a target for therapeutic approaches in inflammatory disorders.
View details for DOI 10.1016/j.redox.2022.102265
View details for PubMedID 35189551
Drp1/Fis1-Dependent Pathologic Fission and Associated Damaged Extracellular Mitochondria Contribute to Macrophage Dysfunction in Endotoxin Tolerance.
Critical care medicine
OBJECTIVES: Recent publications have shown that mitochondrial dynamics can govern the quality and quantity of extracellular mitochondria subsequently impacting immune phenotypes. This study aims to determine if pathologic mitochondrial fission mediated by Drp1/Fis1 interaction impacts extracellular mitochondrial content and macrophage function in sepsis-induced immunoparalysis.DESIGN: Laboratory investigation.SETTING: University laboratory.SUBJECTS: C57BL/6 and BALB/C mice.INTERVENTIONS: Using in vitro and murine models of endotoxin tolerance (ET), we evaluated changes in Drp1/Fis1-dependent pathologic fission and simultaneously measured the quantity and quality of extracellular mitochondria. Next, by priming mouse macrophages with isolated healthy mitochondria (MC) and damaged mitochondria, we determined if damaged extracellular mitochondria are capable of inducing tolerance to subsequent endotoxin challenge. Finally, we determined if inhibition of Drp1/Fis1-mediated pathologic fission abrogates release of damaged extracellular mitochondria and improves macrophage response to subsequent endotoxin challenge.MEASUREMENTS AND MAIN RESULTS: When compared with naive macrophages (NMs), endotoxin-tolerant macrophages (ETM) demonstrated Drp1/Fis1-dependent mitochondrial dysfunction and higher levels of damaged extracellular mitochondria (Mitotracker-Green + events/50 muL: ETM = 2.42 * 106 ± 4,391 vs NM = 5.69 * 105 ± 2,478; p < 0.001). Exposure of NMs to damaged extracellular mitochondria (MH) induced cross-tolerance to subsequent endotoxin challenge, whereas MC had minimal effect (tumor necrosis factor [TNF]-alpha [pg/mL]: NM = 668 ± 3, NM + MH = 221 ± 15, and NM + Mc = 881 ± 15; p < 0.0001). Inhibiting Drp1/Fis1-dependent mitochondrial fission using heptapeptide (P110), a selective inhibitor of Drp1/Fis1 interaction, improved extracellular mitochondrial function (extracellular mitochondrial membrane potential, JC-1 [R/G] ETM = 7 ± 0.5 vs ETM + P110 = 19 ± 2.0; p < 0.001) and subsequently improved immune response in ETMs (TNF-alpha [pg/mL]; ETM = 149 ± 1 vs ETM + P110 = 1,150 ± 4; p < 0.0001). Similarly, P110-treated endotoxin tolerant mice had lower amounts of damaged extracellular mitochondria in plasma (represented by higher extracellular mitochondrial membrane potential, TMRM/MT-G: endotoxin tolerant [ET] = 0.04 ± 0.02 vs ET + P110 = 0.21 ± 0.02; p = 0.03) and improved immune response to subsequent endotoxin treatment as well as cecal ligation and puncture.CONCLUSIONS: Inhibition of Drp1/Fis1-dependent mitochondrial fragmentation improved macrophage function and immune response in both in vitro and in vivo models of ET. This benefit is mediated, at least in part, by decreasing the release of damaged extracellular mitochondria, which contributes to endotoxin cross-tolerance. Altogether, these data suggest that alterations in mitochondrial dynamics may play an important role in sepsis-induced immunoparalysis.
View details for DOI 10.1097/CCM.0000000000005437
View details for PubMedID 35067534
deltaPKC-Mediated Drp1 Phosphorylation Impacts Macrophage Mitochondrial Function and Inflammatory Response to Endotoxin.
Shock (Augusta, Ga.)
BACKGROUND: Recent studies have demonstrated that alterations in mitochondrial dynamics can impact innate immune function. However, the upstream mechanisms which link mitochondrial dynamics to innate immune phenotypes have not been completely elucidated. This study asks if deltaPKC-mediated phosphorylation of Drp1, a key driver of mitochondrial fission, impacts macrophage pro-inflammatory response following bacterial-derived lipopolysaccharide (LPS) stimulation.METHODS: Using RAW 264.7 cells, bone marrow-derived macrophages from C57BL/6J mice, as well as human monocyte-derived macrophages, we first characterized changes in deltaPKC-mediated phosphorylation of Drp1 following LPS stimulation. Next, using rationally-designed peptides that inhibit deltaPKC activation (deltaV1-1) and deltaPKC-Drp1 interaction (psiDrp1), we determined whether deltaPKC-mediated phosphorylation of Drp1 impacts LPS-induced changes in mitochondrial morphology, mitochondrial function, and inflammatory response.RESULTS: Our results demonstrated that deltaPKC-dependent Drp1 activation is associated with increased mitochondrial fission, impaired cellular respiration, and increased mitochondrial reactive oxygen species in LPS-treated macrophages. This is reversed using a rationally-designed peptide which selectively inhibits deltaPKC phosphorylation of Drp1 (psiDrp1). Interestingly, limiting excessive mitochondrial fission using psiDrp1 reduced LPS-triggered pro-inflammatory response, including a decrease in NF-kappaB nuclear localization, decreased iNOS induction, and a reduction in pro-inflammatory cytokines (IL-1beta, TNFalpha, IL-6).CONCLUSION: These data suggest that inhibiting Drp1 phosphorylation by deltaPKC abates the excessive mitochondrial fragmentation and mitochondrial dysfunction that is seen following LPS treatment. Furthermore, these data suggest that limiting deltaPKC-dependent Drp1 activation decreases the pro-inflammatory response following LPS treatment. Altogether, deltaPKC-dependent Drp1 phosphorylation might be an upstream mechanistic link between alterations in mitochondrial dynamics and innate immune phenotypes, and may have therapeutic potential.
View details for DOI 10.1097/SHK.0000000000001885
View details for PubMedID 34738957