Melanie J. Scott

Hemorrhagic Shock Activation of NLRP3 Inflammasome in Lung Endothelial Cells

Hemorrhagic shock (HS) due to major trauma and surgery predisposes the host to the development of systemic inflammatory response syndrome (SIRS), including acute lung injury (ALI), through activating and exaggerating the innate immune response. IL-1β is a crucial proinflammatory cytokine that contributes to the development of SIRS and ALI. Lung endothelial cells (EC) are one important source of IL-1β, and the production of active IL-1β is controlled by the inflammasome. In this study, we addressed the mechanism underlying HS activation of the inflammasome in lung EC. We show that high mobility group box 1 acting through TLR4, and a synergistic collaboration with TLR2 and receptor for advanced glycation end products signaling, mediates HS-induced activation of EC NAD(P)H oxidase. In turn, reactive oxygen species derived from NAD(P)H oxidase promote the association of thioredoxin-interacting protein with the nucleotide-binding oligomerization domain-like receptor protein NLRP3 and subsequently induce inflammasome activation and IL-1β secretion from the EC. We also show that neutrophil-derived reactive oxygen species play a role in enhancing EC NAD(P)H oxidase activation and therefore an amplified inflammasome activation in response to HS. The present study explores a novel mechanism underlying HS activation of EC inflammasome and thus presents a potential therapeutic target for SIRS and ALI induced after HS.

Systemic inflammation and end organ damage following trauma involves functional TLR4 signaling in both bone marrow-derived cells and parenchymal cells

Endogenous damage‐associated molecular pattern (DAMP) molecules are released from cells during traumatic injury, allowing them to interact with pattern recognition receptors such as the toll‐like receptors (TLRs) on other cells and subsequently, to stimulate inflammatory signaling. TLR4, in particular, plays a key role in systemic and remote organ responses to hemorrhagic shock (HS) and peripheral tissue injury in the form of bilateral femur fracture. TLR4 chimeric mice were generated to investigate the cell lineage in which functional TLR4 is needed to initiate the injury response to trauma. Chimeric mice were generated by adoptive bone marrow (BM) transfer, whereby donor marrow was given to an irradiated host using reciprocal combinations of TLR4 wild‐type (WT; C3H/HeOuJ) and TLR4 mutant (Mu; C3H/HeJ) mice. After a period of engraftment, chimeric mice were then subjected to HS or bilateral femur fracture. Control groups, including TLR4‐WT mice receiving WT BM and TLR4‐Mu mice receiving Mu BM, responded to injury in a similar pattern to unaltered HeOuJ and HeJ mice, and protection was afforded to those mice lacking functional TLR4. In contrast, TLR4‐WT mice receiving Mu BM and TLR4‐Mu mice receiving WT BM demonstrated intermediate inflammatory and cellular damage profiles. These data demonstrate that functional TLR4 is required in BM‐derived cells and parenchymal cells for an optimal inflammatory response to trauma.

Lipopolysaccharide Clearance, Bacterial Clearance, and Systemic Inflammatory Responses Are Regulated by Cell Type–Specific Functions of TLR4 during 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.

Endotoxin uptake in mouse liver is blocked by endotoxin pretreatment through a suppressor of cytokine signaling‐1–dependent mechanism

The liver is the main organ that clears lipopolysaccharide (LPS) and hepatocytes are a major cell‐type involved in LPS uptake. LPS tolerance, or desensitization, is important in negative regulation of responses to LPS, but little is known about its mechanisms in hepatocytes. Primary isolated C57BL/6 hepatocytes, and liver in vivo, internalized fluorescent LPS, and this was dependent on Toll‐like receptor 4 (TLR4) at the cell surface but not on TLR4‐TIR signaling through MyD88. LPS clearance from plasma was also TLR4‐dependent. Pretreatment of C57BL/6 hepatocytes with LPS prevented uptake of LPS 24 hours later and this LPS‐mediated suppression was dependent on TLR4 signaling through MyD88. Many regulators of TLR4 signaling have been identified and implicated in LPS desensitization, including suppressor of cytokine signaling 1 (SOCS1). SOCS1 mRNA and protein expression increased after LPS stimulation in hepatocytes and in whole liver. LPS uptake in hepatocytes and liver was significantly reduced following infection with adenoviral vectors overexpressing SOCS1. Similarly, inhibition of SOCS1 using small interfering (si)RNA‐mediated knockdown prevented LPS desensitization in hepatocytes. SOCS1 is known to interact with Toll/IL‐1 receptor associated protein (TIRAP) and cause TIRAP ubiquitination and degradation, which regulates TLR signaling. We have also shown previously that TIRAP regulates LPS uptake in hepatocytes. SOCS1 coimmunoprecipitated with TIRAP in wild type hepatocyte cell lysates up to 8 hours after LPS stimulation, but not at later times. In the same samples, ubiquitinated TIRAP was detected after 4 hours and up to 8 hours after LPS stimulation, but not at later times. Conclusion: These data indicate hepatocytes are desensitized by LPS in a TLR4 signaling‐dependent manner. LPS‐induced SOCS1 upregulation increases degradation of TIRAP and prevents subsequent LPS uptake. The exploitation of these mechanisms of LPS desensitization in the liver may be important in future sepsis therapies. (HEPATOLOGY 2009.)

Hepatocytes express functional NOD1 and NOD2 receptors: A role for NOD1 in hepatocyte CC and CXC chemokine production

BACKGROUND & AIMS NOD-like receptors are recently described cytosolic pattern recognition receptors. NOD1 and NOD2 are members of this family that recognize bacterial cell wall components, diaminopimelic acid and muramyl dipeptide, respectively. Both NOD1 and NOD2 have been associated with many inflammatory diseases, although their role in liver inflammation and infection has not been well studied. METHODS We investigated the role of NOD receptors in mouse liver by assessing expression and activation of NOD1 and NOD2 in liver and primary isolated hepatocytes from C57BL/6 mice. RESULTS Both NOD1 and NOD2 mRNA and protein were highly expressed in hepatocytes and liver. RIP2, the main signaling partner for NODs, was also expressed. Stimulation of hepatocytes with NOD1 ligand (C12-iEDAP) induced NFkappaB activation, activation of MAP kinases and expression of chemokines CCL5 (RANTES) and CXCL1 (KC). C12-iEDAP also synergized with interferon (IFN)gamma to increase iNOS expression and production of nitric oxide. Despite activating NFkappaB, NOD1 ligand did not upregulate hepatocyte production of the acute phase proteins lipopolysaccharide binding protein, serum amyloid A, or soluble CD14 in cell culture supernatants, or upregulate mRNA expression of lipopolysaccharide binding protein, serum amyloid A, C-reactive protein, or serum amyloid P. NOD2 ligand (MDP) did not activate hepatocytes when given alone, but did synergize with Toll-like receptor ligands, lipopolysaccharide (LPS), and polyI:C to activate NFkappaB and MAPK. CONCLUSIONS All together these data suggest an important role for hepatocyte NOD1 in attracting leukocytes to the liver during infection and for hepatic NLRs to augment innate immune responses to pathogens.

Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

MDSC-derived nitric oxide supports the development of Th17 cells in ovarian cancer dependent on the induction of endogenous NOS2 and the cGMP–cGK pathway in Th17 cells.

Macrophage endocytosis of high-mobility group box 1 triggers pyroptosis

Macrophages can be activated and regulated by high-mobility group box 1 (HMGB1), a highly conserved nuclear protein. Inflammatory functions of HMGB1 are mediated by binding to cell surface receptors, including the receptor for advanced glycation end products (RAGE), Toll-like receptor (TLR)2, TLR4, and TLR9. Pyroptosis is a caspase-1-dependent programmed cell death, which features rapid plasma membrane rupture, DNA fragmentation, and release of proinflammatory intracellular contents. Pyroptosis can be triggered by various stimuli, however, the mechanism underlying pyroptosis remains unclear. In this study, we identify a novel pathway of HMGB1-induced macrophage pyroptosis. We demonstrate that HMGB1, acting through RAGE and dynamin-dependent signaling, initiates HMGB1endocytosis, which in turn induces cell pyroptosis. The endocytosis of HMGB1 triggers a cascade of molecular events, including cathepsin B release from ruptured lysosomes followed by pyroptosome formation and caspase-1 activation. We further confirm that HMGB1-induced macrophage pyroptosis also occurs in vivo during endotoxemia, suggesting a pathophysiological significance for this form of pyroptosis in the development of inflammation. These findings shed light on the regulatory role of ligand-receptor internalization in directing cell fate, which may have an important role in the progress of inflammation following infection and injury.

CD40–CD154 interactions between macrophages and natural killer cells during sepsis are critical for macrophage activation and are not interferon gamma dependent

Natural killer (NK) cell interactions with macrophages have been shown to be important during bacterial sepsis in activating macrophages to improve bacterial clearance. The mechanism for this increased activation, however, is unclear. This study determines the relative roles of interferon (IFN)‐γ and CD40/CD154 direct cell interactions on macrophage and NK cell activation in an experimental model of sepsis. Splenic NK cells and peritoneal macrophages were isolated and cultured alone or in coculture, with and without LPS. CD69 expression on NK cells, phagocytosis ability of macrophages, and cell cytokine production was assessed at 24 and 48 h. Coculture of NK cells and macrophages significantly increased activation levels of both cell types, and through experiments culturing NK cells with supernatants from stimulated macrophages and macrophages with supernatants from stimulated NK cells, this activation was determined to be cell‐contact‐dependent. Similar experiments were conducted using NK cells from IFN‐γ deficient (–/–) mice, as well as anti‐IFN‐γ neutralizing antibody. These experiments determined that IFN‐γ is not required for NK or macrophage activation, although it did augment activation levels. Experiments were again repeated using peritoneal macrophages from CD40‐/– mice or splenic NK cells from CD154‐/– mice. CD40/CD154 interactions were important in the ingestion of bacteria by macrophages, but did not affect NK cell activation at 24 h. There was, however, a protective effect of CD40/CD154 interactions on NK cell activation‐induced cell death that occurred at 48 h. CD40/CD154 interactions between macrophages and NK cells are therefore important in macrophage phagocytosis, and are not dependent on IFN‐γ.

Caspase-1 is hepatoprotective during trauma and hemorrhagic shock by reducing liver injury and inflammation.

Adaptive immune responses are induced in liver after major stresses such as hemorrhagic shock (HS) and trauma. There is emerging evidence that the inflammasome, the multiprotein platform that induces caspase-1 activation and promotes interleukin (IL)-1β and IL-18 processing, is activated in response to cellular oxidative stress, such as after hypoxia, ischemia and HS. Additionally, damage-associated molecular patterns, such as those released after injury, have been shown to activate the inflammasome and caspase-1 through the NOD-like receptor (NLR) NLRP3. However, the role of the inflammasome in organ injury after HS and trauma is unknown. We therefore investigated inflammatory responses and end-organ injury in wild-type (WT) and caspase-1−/− mice in our model of HS with bilateral femur fracture (HS/BFF). We found that caspase-1−/− mice had higher levels of systemic inflammatory cytokines than WT mice. This result corresponded to higher levels of liver damage, cell death and neutrophil influx in caspase-1−/− liver compared with WT, although there was no difference in lung damage between experimental groups. To determine if hepatoprotection also depended on NLRP3, we subjected NLRP3−/− mice to HS/BFF, but found inflammatory responses and liver damage in these mice was similar to WT. Hepatoprotection was also not due to caspase-1-dependent cytokines, IL-1β and IL-18. Altogether, these data suggest that caspase-1 is hepatoprotective, in part through regulation of cell death pathways in the liver after major trauma, and that caspase-1 activation after HS/BFF does not depend on NLRP3. These findings may have implications for the treatment of trauma patients and may lead to progress in prevention or treatment of multiple organ failure (MOF).

Hepatocytes enhance effects of lipopolysaccharide on liver nonparenchymal cells through close cell interactions.

The response to lipopolysaccharide (LPS) in the liver is complex, requiring cell-to-cell interactions between hepatocytes and liver nonparenchymal cells (NPC), in particular, Kupffer cells. Previous studies show that cytokines produced by Kupffer cells stimulated with LPS can, in turn, activate hepatocytes. In the present study, we sought to examine whether the reverse, hepatocyte (HC)-NPC interactions, is important in cytokine production in mixed cell cocultures. LPS-stimulated production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 from NPC was augmented in mixed HC-NPC cocultures, as compared with NPC monocultures. This HC-NPC interaction was not observed when hepatocytes were cocultured with NPC from TLR4-mutant (C3H/HeJ) mice or CD14-deficient mice. The effect was partially lost when hepatocytes from lipopolysaccharide-binding protein (LBP)-deficient mice were cocultured with wild-type mice. These data indicate that functional TLR4 and CD14 are required for NPC production of cytokines and that at least one of the critical components from hepatocytes is LBP. The augmented cytokine production by mixed HC-NPC cocultures was abrogated when the cells were separated by a filter system, indicating that close cell interactions are also required for this interaction. Thus, interaction between hepatocytes and NPC are critical for cytokine secretion by NPC.