| Abstract: | Extracellular high-mobility group box (HMGB)1 mediates inflammation during sterile and infectious injury and contributes importantly to disease pathogenesis. The first critical step in the release of HMGB1 from activated immune cells is mobilization from the nucleus to the cytoplasm, a process dependent upon hyperacetylation within two HMGB1 nuclear localization sequence (NLS) sites. The inflammasomes mediate the release of cytoplasmic HMGB1 in activated immune cells, but the mechanism of HMGB1 translocation from nucleus to cytoplasm was previously unknown. Here, we show that pharmacological inhibition of JAK/STAT1 inhibits LPS-induced HMGB1 nuclear translocation. Conversely, activation of JAK/STAT1 by type 1 interferon (IFN) stimulation induces HMGB1 translocation from nucleus to cytoplasm. Mass spectrometric analysis unequivocally revealed that pharmacological inhibition of the JAK/STAT1 pathway or genetic deletion of STAT1 abrogated LPS- or type 1 IFN-induced HMGB1 acetylation within the NLS sites. Together, these results identify a critical role of the JAK/STAT1 pathway in mediating HMGB1 cytoplasmic accumulation for subsequent release, suggesting that the JAK/STAT1 pathway is a potential drug target for inhibiting HMGB1 release.High-mobility group box 1 (HMGB1), a ubiquitous DNA-binding protein, is a promiscuous sensor driving nucleic acid-mediated immune responses and a pathogenic inflammatory mediator in sepsis, arthritis, colitis, and other disease syndromes (1–5). Immune cells actively release HMGB1 after activation by exposure to pathogen-associated molecular patterns or damage-associated molecular patterns, including lipopolysaccharide (LPS) and inflammasome agonists (1, 6, 7). High levels of extracellular HMGB1 accumulate in patients with infectious and sterile inflammatory diseases. Extracellular disulfide HMGB1 stimulates macrophages to release TNF and other inflammatory mediators by binding and signaling through Toll-like receptor (TLR)4. Reduced HMGB1 facilitates immune cell migration by interacting with the receptor for advanced glycation end products (RAGE) and CXCL12 (8–12), a process regulated by posttranslational redox-dependent mechanisms. Administration of neutralizing anti-HMGB1 mAbs or other HMGB1 antagonists significantly reduces the severity of inflammatory disease, promotes bacterial clearance during Pseudomonas aeruginosa or Salmonella typhimurium infection and attenuates memory impairment in sepsis survivors (1, 13–15). Together, these and other findings indicate the importance of a mechanistic understanding of HMGB1 release from activated immune cells and the regulatory signaling pathways controlling these processes.Most cytokines harbor a leader peptide that facilitates secretion through the endoplasmic reticulum–Golgi exocytotic route. HMGB1, which lacks a leader peptide, is released via unconventional protein secretion pathways (1, 6, 7). In quiescent cells, most HMGB1 is localized in the nucleus. Upon activation of immune cells, efficient HMGB1 release requires acetylation of HMGB1 within the two nuclear localization sequence (NLS) sites and subsequent HMGB1 accumulation in the cytoplasm (1, 6, 16–20). HMGB1 release is mediated by inflammasome activation during pyroptosis, a form of proinflammatory programmed cell death (6, 7, 22–24). Protein kinase (PK)R is a critical regulator of inflammasome-dependent HMGB1 release (6, 25). Pharmacological inhibition of PKR abrogates LPS-induced HMGB1 release by macrophages but does not prevent nuclear translocation of HMGB1 to cytoplasm. This suggests that some other, as yet unknown, inflammasome-independent pathway regulates HMGB1 translocation from nucleus to cytoplasm.We and others have previously established an important role of type 1 and type 2 interferons (IFNs) and downstream JAK/STAT1 signaling activation in mediating HMGB1 release (26–28). Pharmacological inhibition of JAK/STAT, genetic deletion of STAT1, or inhibition of extracellular IFN-β with neutralizing antibodies significantly abrogates LPS-induced HMGB1 release from macrophages (26–28). Importantly, pharmacological inhibition of the JAK/STAT1 pathway, genetic deletion of STAT1, or inhibition of IFN-β expression by genetic deletion of IRF3 significantly promotes survival in both lethal endotoxemia and experimental sepsis (28–30). Accordingly, we reasoned here that JAK/STAT1 may represent a critical signaling mechanism controlling HMGB1 translocation from nucleus to cytoplasm. |
