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HMGB1 redox during sepsis.

ABSTRACT: During sepsis, the alarmin HMGB1 is released from tissues and promotes systemic inflammation that results in multi-organ damage, with the kidney particularly susceptible to injury. The severity of inflammation and pro-damage signaling mediated by HMGB1 appears to be dependent on the alarmin's redox state. Therefore, we examined HMGB1 redox in kidney cells during sepsis. Using intravital microscopy, CellROX labeling of kidneys in live mice indicated increased ROS generation in the kidney perivascular endothelium and tubules during lipopolysaccharide (LPS)-induced sepsis. Subsequent CellROX and MitoSOX labeling of LPS-stressed endothelial and kidney proximal tubule cells demonstrated increased ROS generation in these cells as sepsis worsens. Consequently, HMGB1 oxidation increased in the cytoplasm of kidney cells during its translocation from the nucleus to the circulation, with the degree of oxidation dependent on the severity of sepsis, as measured in in vivo mouse samples using a thiol assay and mass spectrometry (LC-MS/MS). The greater the oxidation of HMGB1, the greater the ability of the alarmin to stimulate pro-inflammatory cyto-/chemokine release (measured by Luminex Multiplex) and alter mitochondrial ATP generation (Luminescent ATP Detection Assay). Administration of glutathione and thioredoxin inhibitors to cell cultures enhanced HMGB1 oxidation during sepsis in endothelial and proximal tubule cells, respectively. In conclusion, as sepsis worsens, ROS generation and HMGB1 oxidation increases in kidney cells, which enhances HMGB1's pro-inflammatory signaling. Conversely, the glutathione and thioredoxin systems work to maintain the protein in its reduced state.

SUBMITTER: Abdulmahdi W

PROVIDER: S-EPMC5554965 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Oxidative mitochondrial damage is closely linked to inflammation and cell death, but low levels of reactive oxygen and nitrogen species serve as signals that involve mitochondrial repair and resolution of inflammation. More specifically, cytoprotection relies on the elimination of damaged mitochondria by selective autophagy (mitophagy) during mitochondrial quality control. This aim of this study was to identify and localize mitophagy in the mouse lung as a potentially upregulatable redox response to Staphylococcus aureus sepsis. Fibrin clots loaded with S. aureus (1×10(7) CFU) were implanted abdominally into anesthetized C57BL/6 and B6.129X1-Nfe2l2tm1Ywk/J (Nrf2(-/-)) mice. At the time of implantation, mice were given vancomycin (6mg/kg) and fluid resuscitation. Mouse lungs were harvested at 0, 6, 24, and 48h for bronchoalveolar lavage (BAL), Western blot analysis, and qRT-PCR. To localize mitochondria with autophagy protein LC3, we used lung immunofluorescence staining in LC3-GFP transgenic mice. In C57BL/6 mice, sepsis-induced pulmonary inflammation was detected by significant increases in mRNA for the inflammatory markers IL-1β and TNF-α at 6 and 24h, respectively. BAL cell count and protein also increased. Sepsis suppressed lung Beclin-1 protein, but not mRNA, suggesting activation of canonical autophagy. Notably sepsis also increased the LC3-II autophagosome marker, as well as the lung׳s noncanonical autophagy pathway as evidenced by loss of p62, a redox-regulated scaffolding protein of the autophagosome. In LC3-GFP mouse lungs, immunofluorescence staining showed colocalization of LC3-II to mitochondria, mainly in type 2 epithelium and alveolar macrophages. In contrast, marked accumulation of p62, as well as attenuation of LC3-II in Nrf2-knockout mice supported an overall decrease in autophagic turnover. The downregulation of canonical autophagy during sepsis may contribute to lung inflammation, whereas the switch to noncanonical autophagy selectively removes damaged mitochondria and accompanies tissue repair and cell survival. Furthermore, mitophagy in the alveolar region appears to depend on activation of Nrf2. Thus, efforts to promote mitophagy may be a useful therapeutic adjunct for acute lung injury in sepsis.

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HMGB1 redox during sepsis.

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