Roop Gill

Linking oxidative stress to inflammation: Toll-like receptors

Injury caused by oxidative stress occurs in many clinical scenarios involving ischemia and reperfusion such as organ transplantation, hemorrhagic shock (HS), myocardial infarction, and cerebral vascular accidents. Activation of the immune system as a result of disturbances in the redox state of cells seems to contribute to tissue and organ damage in these conditions. The link between oxidative stress and inflammatory pathways is poorly understood. Recently, Toll-like receptors (TLRs) have been shown to mediate the inflammatory response seen in experimental ischemia and reperfusion (I/R). The TLR family of receptors involved in alerting the innate immune system of danger seems to be activated by damage-associated molecular pattern molecules (DAMPs) that are released during conditions of oxidative stress. In this review, we examine the role of TLRs in various experimental models of oxidative stress such as HS and I/R. We also report on potential DAMPs that may interact with TLRs in mediating injury. Finally, potential mechanisms by which reactive oxygen species from NADPH oxidase can signal the commencement of inflammatory pathways through TLRs are explored.

Systemic inflammation and liver injury following hemorrhagic shock and peripheral tissue trauma involve functional TLR9 signaling on bone marrow-derived cells and parenchymal cells.

Hemorrhagic shock due to trauma (HS/T) induces an inflammatory response that can contribute to end-organ injury. The pathways involved in the initiation and propagation of HS/T-induced inflammation are incompletely understood. Here, we hypothesized that the DNA sensor TLR9 would have a role in inflammatory signaling after HS/T. Using mice expressing a nonfunctional, mutant form of TLR9, we identified a role of TLR9 in driving the initial cytokine response and liver damage in a model of hemorrhagic shock and bilateral femur fracture. Circulating DNA levels were found to correlate with the degree of tissue damage. Experiments using chimeric mice show that TLR9 on both bone marrow-derived cells and parenchymal cells are important for the TLR9-mediated liver and tissue damage, as well as systemic inflammation after HS/T. These data suggest that release of DNA may be a driver of the inflammatory response to severe injury as well as a marker of the extent of tissue damage. One of the sensors of DNA in the setting of HS/T seems to be TLR9.ABBREVIATIONS-TLRs-Toll-like receptors; HS-hemorrhagic shock; HS/T-hemorrhagic shock + bilateral femur fracture; ALT-alanine aminotransferase; AST-aspartate aminotransferase; ssDNA-single-stranded DNA; dsDNA-double-stranded DNA

DNA attenuates enterocyte Toll-like receptor 4-mediated intestinal mucosal injury after remote trauma

Intestinal mucosal injury occurs after remote trauma although the mechanisms that sense remote injury and lead to intestinal epithelial disruption remain incompletely understood. We now hypothesize that Toll-like receptor 4 (TLR4) signaling on enterocytes after remote injury, potentially through the endogenous TLR4 ligand high-mobility group box-1 (HMGB1), could lead to intestinal dysfunction and bacterial translocation and that activation of TLR9 with DNA could reverse these effects. In support of this hypothesis, exposure of TLR4-expressing mice to bilateral femur fracture and systemic hypotension resulted in increased TLR4 expression and signaling and disruption of the ileal mucosa, leading to bacterial translocation, which was not observed in TLR4-mutant mice. TLR4 signaling in enterocytes, not immune cells, was required for this effect, as adenoviral-mediated inhibition of TLR4 in enterocytes prevented these findings. In seeking to identify the endogenous TLR4 ligands involved, the expression of HMGB1 was increased in the intestinal mucosa after injury in wild-type, but not TLR4-mutant, mice, and administration of anti-HMGB1 antibodies reduced both intestinal mucosal TLR4 signaling and bacterial translocation after remote trauma. Strikingly, mucosal injury was significantly increased in TLR9-mutant mice, whereas administration of exogenous DNA reduced the extent of TLR4-mediated enterocyte apoptosis, restored mucosal healing, and maintained the histological integrity of the intestinal barrier after remote injury. Taken together, these findings identify a novel link between remote injury and enterocyte TLR4 signaling leading to barrier injury, potentially through HMGB1 as a ligand, and demonstrate the reversal of these adverse effects through activation of TLR9.

Pivotal Advance: The pattern recognition receptor ligands lipopolysaccharide and polyinosine-polycytidylic acid stimulate factor B synthesis by the macrophage through distinct but overlapping mechanisms

TLRs and complement are critical to the host response in sepsis, trauma, and ischemia/reperfusion. We hypothesize that TLR stimulation leads to synthesis and release of complement components by macrophages, an important source of extrahepatic complement. RAW264.7 macrophages or peritoneal macrophages from WT and TLR4‐, TLR3‐, TRIF‐, or MyD88‐deficient mice were cultured under standard conditions. In some experiments, cells were pretreated with inhibitors of MAPKs or a NF‐κB inhibitor. Cells were stimulated with TLR ligands at known stimulatory concentrations. Intratracheal and i.p. injections were also performed in mice. RT‐PCR, Western blotting, and immunocytochemistry were used for analysis. Using a RT‐PCR‐based panel, we demonstrate that of 18 complement components tested, factor B of the alternative pathway is the most robustly up‐regulated complement component in macrophages in response to LPS. This up‐regulation results in release of factor B into the media. Up‐regulation of factor B by LPS is dependent on TLR4, TRIF, JNK, and NF‐κB. A screen of other TLR ligands demonstrated that stimulation with poly I:C (dsRNA analog) also results in up‐regulation of factor B, which is dependent on JNK and NF‐κB but independent of TLR3 and TRIF. Up‐regulation of factor B is also observed after intratracheal and i.p. injection of LPS or poly I:C in vivo. PRR stimulation profoundly influences production and release of factor B by macrophages. Understanding the mechanisms of PRR‐mediated complement production may lead to strategies aimed at preventing tissue damage in diverse settings, including sepsis, trauma, and ischemia/reperfusion.

Complement factor 3 deficiency attenuates hemorrhagic shock-related hepatic injury and systemic inflammatory response syndrome

Although complement activation is known to occur in the setting of severe hemorrhagic shock and tissue trauma (HS/T), the extent to which complement drives the initial inflammatory response and end-organ damage is uncertain. In this study, complement factor 3-deficient (C3(-/-)) mice and wild-type control mice were subjected to 1.5-h hemorrhagic shock, bilateral femur fracture, and soft tissue injury, followed by 4.5-h resuscitation (HS/T). C57BL/6 mice were also given 15 U of cobra venom factor (CVF) or phosphate-buffered saline injected intraperitoneally, followed by HS/T 24 h later. The results showed that HS/T resulted in C3 consumption in wild-type mice and C3 deposition in injured livers. C3(-/-) mice had significantly lower serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and circulating DNA levels, together with much lower circulating interleukin (IL)-6, IL-10, and high-mobility group box 1 (HMGB1) levels. Temporary C3 depletion by CVF preconditioning also led to reduced transaminases and a blunted cytokine release. C3(-/-) mice displayed well-preserved hepatic structure. C3(-/-) mice subjected to HS/T had higher levels of heme oxygenase-1, which has been associated with tissue protection in HS models. Our data indicate that complement activation contributes to inflammatory pathways and liver damage in HS/T. This suggests that targeting complement activation in the setting of severe injury could be useful.

Management of peripheral vascular disease compromising renal allograft placement and function: review of the literature with an illustrative case

Gill R, Shapiro R, Kayler LK. Management of peripheral vascular disease compromising renal allograft placement and function: review of the literature with an illustrative case.
Clin Transplant 2011: 25: 337–344.

TLR9 deficiency in parenchymal or immune cells is associated with reduced tissue damage in a trauma model

Temp (C) 0.2 0.0 0.2 0.1 0.7 0.4 0.8 0.3 1.6 0.1 1.6 0.2 HR (bpm) 4 39 11 4 92 3 7 42 1 35 5 WBCs 1 0.9 2 13 0.4 4 0.7 5 1.3 6 0.8 LF/HF 0.9 0.36 0.54 0.41 2.25 1.70 1.9 0.95 2.03 1.53 5.18 0.92 TNF (pg/mL) 6 09 49 32 5 17 244 22 191 64 IL-6 (pg/mL) 15 12 4 02 2 19 77 35 323 46 320 28 Significant vs placebo. CONCLUSIONS: Both vital sign changes and HRV markers of autonomic imbalance were seen at LPS doses 2 to 4 times lower than those required for significant elevations in TNF, IL-6, and WBC. This finding suggests that endotoxinemia is associated with changes in autonomic balance well before overt acute inflammation arises. Furthermore, it suggests the possibility of unidentified signaling mechanisms that partially precede proinflammatory cytokines as the causal agents of early vital sign and autonomic changes.

Hepatic dysfunction after shock: Clinical parameters and biological pathways for therapeutic intervention*

The prevalence of hepatic dysfunction was thought to be ≈ 1% in intensive care unit (ICU) patients, but is now being recognized to be > 10%. Mortality and length of ICU stay are directly affected by hepatic dysfunction, raising the need for a better understanding of this disorder in order to develop therapeutic options that go beyond the current practice of supportive care. The etiologies of hypoxic hepatitis are discussed, together with their differentiating physiological parameters. The livers compensatory mechanisms are examined at the level of the microcirculation. The cytokine milieu and cells responsible are considered. Survival versus cell death by means of necrosis, apoptosis, or autophagy is determined by the interplay of intracellular pathways. Specific pathways discussed involve reactive oxygen species, Toll-like receptor 4, heme oxygenase, transcription factors such as nuclear factor-κB, mitogen-activated protein kinase and protein kinase B. From this basis, current and future therapeutic str...