Summary Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.

The morbidity associated with bacterial sepsis is the result of host immune responses to pathogens, which are dependent on pathogen recognition by pattern recognition receptors, such as TLR4. TLR4 is expressed on a range of cell types, yet the mechanisms by which cell-specific functions of TLR4 lead to an integrated sepsis response are poorly understood. To address this, we generated mice in which TLR4 was specifically deleted from myeloid cells (LysMTLR4KO) or hepatocytes (HCTLR4KO) and then determined survival, bacterial counts, host inflammatory responses, and organ injury in a model of cecal ligation and puncture (CLP), with or without antibiotics. LysM-TLR4 was required for phagocytosis and efficient bacterial clearance in the absence of antibiotics. Survival, the magnitude of the systemic and local inflammatory responses, and liver damage were associated with bacterial levels. HCTLR4 was required for efficient LPS clearance from the circulation, and deletion of HCTLR4 was associated with enhanced macrophage phagocytosis, lower bacterial levels, and improved survival in CLP without antibiotics. Antibiotic administration during CLP revealed an important role for hepatocyte LPS clearance in limiting sepsis-induced inflammation and organ injury. Our work defines cell type–selective roles for TLR4 in coordinating complex immune responses to bacterial sepsis and suggests that future strategies for modulating microbial molecule recognition should account for varying roles of pattern recognition receptors in multiple cell populations.

BaCKgRoUND aND aIMS: Itaconate, a metabolite of the tricarboxylic acid cycle, plays anti-inflammatory roles in macrophages during endotoxemia. The mechanisms underlying its anti-inflammatory roles have been shown to be mediated by the modulation of oxidative stress, an important mechanism of hepatic ischemia-reperfusion (I/R) injury. However, the role of itaconate in liver I/R injury is unknown. appRoaCH aND ReSUltS: We found that deletion of immune-responsive gene 1 (IRG1), encoding for the enzyme producing itaconate, exacerbated liver injury and systemic inflammation. Furthermore, bone marrow adoptive transfer experiments indicated that deletion of IRG1 in both hematopoietic and nonhematopoietic compartments contributes to the protection mediated by IRG1 after I/R. Interestingly, the expression of IRG1 was up-regulated in hepatocytes after I/R and hypoxia/reoxygenation-induced oxidative stress. Modulation of the IRG1 expression levels in hepatocytes regulated hepatocyte cell death. Importantly, addition of 4-octyl itaconate significantly improved liver injury and hepatocyte cell death after I/R. Furthermore, our data indicated that nuclear factor erythroid 2-related factor 2 (Nrf2) is required for the protective effect of IRG1 on mouse and human hepatocytes against oxidative stress-induced injury. Our studies document the important role of IRG1 in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that the IRG1/itaconate pathway activates Nrf2-mediated antioxidative response in hepatocytes to protect liver from I/R injury. CoNClUSIoNS: Our data expand on the importance of IRG1/itaconate in nonimmune cells and identify itaconate as a potential therapeutic strategy for this unfavorable postsurgical complication.

SignificanceCD24+CD133+ liver cancer stem cells (LCSCs) express higher levels of the inducible nitric oxide synthase (iNOS) and possess self-renewal and tumor growth properties. iNOS is associated with more aggressive hepatocellular carcinoma (HCC), leading to the upregulation of Notch1 signaling. The activation of Notch1 by iNOS/NO is dependent on cGMP/PKG-mediated activation of TACE and upregulation of iRhom-2. The expression of iNOS, CD24, and CD133 correlates with the expression of activated TACE and Notch signaling in more aggressive human HCC. These findings have implications for understanding how LCSCs are regulated in the setting of chronic inflammation, where signals to upregulate iNOS are often present. Targeting iNOS could have therapeutic benefit in HCC.

High mobility group box 1 (HMGB1) is a multifunctional nuclear protein, probably known best as a prototypical alarmin or damage-associated molecular pattern (DAMP) molecule when released from cells. However, HMGB1 has multiple functions that depend on its location in the nucleus, in the cytosol, or extracellularly after either active release from cells, or passive release upon lytic cell death. Movement of HMGB1 between cellular compartments is a dynamic process induced by a variety of cell stresses and disease processes, including sepsis, trauma, and hemorrhagic shock.Location of HMGB1 is intricately linked with its function and is regulated by a series of posttranslational modifications. HMGB1 function is also regulated by the redox status of critical cysteine residues within the protein, and is cell-type dependent. This review highlights some of the mechanisms that contribute to location and functions of HMGB1, and focuses on some recent insights on important intracellular effects of HMGB1 during sepsis and trauma. K E Y W O R D S AIM2, autophagy, caspase-11, DAMPs, pyroptosis 2 NUCLEAR-TO-CYTOPLASM SHUTTLING OF HMGB1 HMGB1 consists of 215 aa residues, and contains 2 HMG DNAbinding domains, designated as A and B boxes, together with a negatively charged C-terminal acidic region. 14 Two nuclear localization sites (NLSs), one located in the A box (aa 28-44) and one in the B box (aa 179-185), control the nuclear localization of HMGB1 under homeostatic states. 14 Posttranslational modifications of NLS sites, including acetylation, phosphorylation, and methylation, regulate the ability of HMGB1 to translocate to the cytoplasm during cellular stress.Once in the cytoplasm HMGB1 can affect multiple inflammatory responses, and can be actively released into the extracellular space and circulation.

Liver ischemia-reperfusion (I/R) injury occurs through induction of oxidative stress and release of damage-associated molecular patterns (DAMPs), including cytosolic DNA released from dysfunctional mitochondria or from the nucleus. Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) is a cytosolic DNA sensor known to trigger stimulator of interferon genes (STING) and downstream type 1 interferon (IFN-I) pathways, which are pivotal innate immune system responses to pathogen. However, little is known about the role of cGAS/STING in liver I/R injury. We subjected C57BL/6 (WT), cGAS knockout (cGAS), and STING-deficient (STING) mice to warm liver I/R injury and that found cGAS mice had significantly increased liver injury compared with WT or STING mice, suggesting a protective effect of cGAS independent of STING. Liver I/R upregulated cGAS in vivo and also in vitro in hepatocytes subjected to anoxia/reoxygenation (A/R). We confirmed a previously published finding that hepatocytes do not express STING under normoxic conditions or after A/R. Hepatocytes and liver from cGAS mice had increased cell death and reduced induction of autophagy under hypoxic conditions as well as increased apoptosis. Protection could be restored in cGAS hepatocytes by overexpression of cGAS or by pretreatment of mice with autophagy inducer rapamycin. Our findings indicate a novel protective role for cGAS in the regulation of autophagy during liver I/R injury that occurs independently of STING. NEW & NOTEWORTHY Our studies are the first to document the important role of cGAS in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that cGAS protects liver from I/R injury in a STING-independent manner.

High-mobility group box-1 (HMGB1), a ubiquitous nuclear protein, acts as a late mediator of lethality when released extracellularly during sepsis. The major source of circulating HMGB1 in sepsis is hepatocytes. However, the mechanism of HMGB1 release of hepatocytes during sepsis is not very clear. We have previously shown that bacterial endotoxin [lipopolysaccharide (LPS)] sensing pathways, including Toll-like receptor (TLR)4 and caspase-11, regulate hepatocyte HMGB1 release in response to LPS. Here, we report the novel function of caspase-11 and gasdermin D (GsdmD) in LPS-induced active HMGB1 released from hepatocytes. HMGB1 release during endotoxemia was caspase-11/GsdmD dependent via an active way in vivo and in vitro. Caspase-11/GsdmD was responsible for HMGB1 translocation from nucleus to the cytoplasm via calcium changing-induced phosphorylation of calcium-calmodulin kinase kinase (camkk)β during endotoxemia. Cleaved GsdmD accumulated on the endoplasmic reticulum, suggesting this may lead to calcium leak and intracellular calcium increase. Furthermore, we investigated that exosome was an important pathway for HMGB1 release from hepatocytes; this process was dependent on TLR4, independent of caspase-11 and GsdmD in vivo and in vitro. These findings provide a novel mechanism that TLR4 signaling results in an increase in caspase-11 expression, as well as increased exosome release, while caspase-11/GsdmD activation/cleavage leads to accumulation of HMGB1 in the cytoplasm through a process associated with the release of calcium from the endoplasmic reticulum and camkkβ activation.

Thrombocytopenia impairs host defense and hemostasis in sepsis. However, the mechanisms of how platelets regulate host defense are not fully understood. High-mobility group box 1 (HMGB1), a danger-associated molecular pattern protein, is released during infection and contributes to the pathogenesis of sepsis. Platelets express HMGB1, which is released on activation and has been shown to play a critical role in thrombosis, monocyte recruitment, and neutrophil extracellular trap (NET) production. However, the contribution of platelet HMGB1 to host defense is unknown. To determine the role of platelet HMGB1 in polymicrobial sepsis, platelet-specific HMGB1 knockout (HMGB1 platelet factor 4 [PF4]) mice were generated and were subjected to cecal ligation and puncture (CLP), a clinically relevant intra-abdominal sepsis model. Compared with HMGB1 Flox mice and wild-type (WT) mice, HMGB1 PF4 mice showed significantly higher bacterial loads in the peritoneum and blood, an exaggerated systemic inflammation response, and significantly greater mortality after CLP. Deletion of HMGB1 in platelets was associated with lower platelet-derived chemokines (PF4 and RANTES) in the peritoneal cavity, and a decrease of platelet-neutrophil interaction in the lung after CLP. In vitro, neutrophils cocultured with activated HMGB1 knockout platelets showed fewer platelet-neutrophil aggregates, reduced reactive oxygen species (ROS) burst as compared with control. Taken together, these data reveal an unrecognized role of platelet HMGB1 in the regulation of neutrophil recruitment and activation via modulation of platelet activation during sepsis.