Cutting edge: high-mobility group box 1 preconditioning protects against liver ischemia-reperfusion injury

High mobility group box 1 (HMGB1) is a NF released extracellularly as a late mediator of lethality in sepsis and as an early mediator of inflammation following injury. Here we demonstrate that in contrast to the proinflammatory role of HMGB1, preconditioning with HMGB1 results in protection following hepatic ischemia/reperfusion (I/R). Pretreatment of mice with HMGB1 significantly decreased liver damage after I/R. The protection observed in mice pretreated with HMGB1 was associated with a higher expression of IL-1R-associated kinase-M, a negative regulator of TLR4 signaling, compared with controls. We thus explored the possibility that HMGB1 preconditioning was mediated through TLR4 activation. HMGB1 preconditioning failed to provide protection in TLR4 mutant (C3H/HeJ) mice, but successfully reduced damage in TLR4 wild-type (C3H/HeOuj) mice. Our studies demonstrate that in contrast to the role of HMGB1 as an early mediator of inflammation and organ damage in hepatic I/R, HMGB1 preconditioning can be protective.     

Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcgammaRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.     

Systemic inflammation and remote organ damage following bilateral femur fracture requires Toll-like receptor 4

Extensive soft tissue injury and bone fracture are significant contributors to the initial systemic inflammatory response in multiply injured patients. Systemic inflammation can lead to organ dysfunction remote from the site of traumatic injury. The mechanisms underlying the recognition of peripheral injury and the subsequent activation of the immune response are unknown. Toll-like receptors (TLRs) recognize microbial products but also may recognize danger signals released from damaged tissues. Here we report that peripheral tissue trauma initiates systemic inflammation and remote organ dysfunction. Moreover, this systemic response to a sterile local injury requires toll-like receptor 4 (TLR4). Compared with wild-type (C3H/HeOuJ) mice, TLR4 mutant (C3H/HeJ) mice demonstrated reduced systemic and hepatic inflammatory responses to bilateral femur fracture. Trauma-induced nuclear factor (NF)-kappaB activation in the liver required functional TLR4 signaling. CD14-/- mice failed to demonstrate protection from fracture-induced systemic inflammation and hepatocellular injury. Therefore, our results also argue against a contribution of intestine-derived LPS to this process. These findings identify a critical role for TLR4 in the rapid recognition and response pathway to severe traumatic injury. Application of these findings in an evolutionary context suggests that multicellular organisms have evolved to use the same pattern recognition receptor for surviving traumatic and infectious challenges.     

An Adequately Robust Early TNF-α Response Is a Hallmark of Survival Following Trauma/Hemorrhage

Background

Trauma/hemorrhagic shock (T/HS) results in cytokine-mediated acute inflammation that is generally considered detrimental.

Methodology/Principal Findings

Paradoxically, plasma levels of the early inflammatory cytokine TNF-α (but not IL-6, IL-10, or NO₂ ^-/NO₃ ^-) were significantly elevated within 6 h post-admission in 19 human trauma survivors vs. 4 non-survivors. Moreover, plasma TNF-α was inversely correlated with Marshall Score, an index of organ dysfunction, both in the 23 patients taken together and in the survivor cohort. Accordingly, we hypothesized that if an early, robust pro-inflammatory response were to be a marker of an appropriate response to injury, then individuals exhibiting such a response would be predisposed to survive. We tested this hypothesis in swine subjected to various experimental paradigms of T/HS. Twenty-three anesthetized pigs were subjected to T/HS (12 HS-only and 11 HS + Thoracotomy; mean arterial pressure of 30 mmHg for 45–90 min) along with surgery-only controls. Plasma obtained at pre-surgery, baseline post-surgery, beginning of HS, and every 15 min thereafter until 75 min (in the HS only group) or 90 min (in the HS + Thoracotomy group) was assayed for TNF-α, IL-6, IL-10, and NO₂ ^-/NO₃ ^-. Mean post-surgery±HS TNF-α levels were significantly higher in the survivors vs. non-survivors, while non-survivors exhibited no measurable change in TNF-α levels over the same interval.

Conclusions/Significance

Contrary to the current dogma, survival in the setting of severe, acute T/HS appears to be associated with an immediate increase in serum TNF-α. It is currently unclear if this response was the cause of this protection, a marker of survival, or both. This abstract won a Young Investigator Travel Award at the SHOCK 2008 meeting in Cologne, Germany.     

Differential regulation by fucoidan of IFN‐γ‐induced NO production in glial cells and macrophages

Fucoidan has shown numerous biological actions; however, the molecular bases of these actions have being issued. We examined the effect of fucoidan on NO production induced by IFN‐γ and the molecular mechanisms underlying these effects in two types of cells including glia (C6, BV‐2) and macrophages (RAW264.7, peritoneal primary cells). Fucoidan affected IFN‐γ‐induced NO and/or iNOS expression both in macrophages and glial cells but in a contrast way. Our data showed that in C6 glioma cells both JAK/STAT and p38 signaling positively regulated IFN‐γ‐induced iNOS, which were inhibited by fucoidan. In contrast, in RAW264.7 cells JAK/STAT is a positive regulator whereas p38 is a negative regulator of NO/iNOS production. In RAW264.7 cells, fucoidan enhanced p38 activation and induced TNF‐α production. We also confirmed the dual regulation of p38 in BV‐2 microglia and primary peritoneal macrophages. From these results, we suggest that fucoidan affects not only IFN‐γ‐induced NO/iNOS production differently in brain and peritoneal macrophages due to the different roles of p38 but the effects on TNF‐α production in the two cell types. These novel observations including selective and cell‐type specific effects of fucoidan on IFN‐γ‐mediated signaling and iNOS expression raise the possibility that it alters the sensitivity of cells to the p38 activation. J. Cell. Biochem. 111: 1337–1345, 2010. © 2010 Wiley‐Liss, Inc.     

Hypoglycemia induced changes in cholinergic receptor expression in the cerebellum of diabetic rats

Glucose homeostasis in humans is an important factor for the functioning of nervous system. Hypoglycemia and hyperglycemia is found to be associated with central and peripheral nerve system dysfunction. Changes in acetylcholine receptors have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). In the present study we showed the effects of insulin induced hypoglycemia and streptozotocin induced diabetes on the cerebellar cholinergic receptors, GLUT3 and muscle cholinergic activity. Results showed enhanced binding parameters and gene expression of Muscarinic M1, M3 receptor subtypes in cerebellum of diabetic (D) and hypoglycemic group (D + IIH and C + IIH). α7nAchR gene expression showed a significant upregulation in diabetic group and showed further upregulated expression in both D + IIH and C + IIH group. AchE expression significantly upregulated in hypoglycemic and diabetic group. ChAT showed downregulation and GLUT3 expression showed a significant upregulation in D + IIH and C + IIH and diabetic group. AchE activity enhanced in the muscle of hypoglycemic and diabetic rats. Our studies demonstrated a functional disturbance in the neuronal glucose transporter GLUT3 in the cerebellum during insulin induced hypoglycemia in diabetic rats. Altered expression of muscarinic M1, M3 and α7nAchR and increased muscle AchE activity in hypoglycemic rats in cerebellum is suggested to cause cognitive and motor dysfunction. Hypoglycemia induced changes in ChAT and AchE gene expression is suggested to cause impaired acetycholine metabolism in the cerebellum. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. The results shows that cerebellar cholinergic neurotransmission is impaired during hyperglycemia and hypoglycemia and the hypoglycemia is causing more prominent imbalance in cholinergic neurotransmission which is suggested to be a cause of cerebellar dysfunction associated with hypoglycemia.     

Up-regulation of alpha-inhibin expression in the fetal ovary of estrogen-suppressed baboons is associated with impaired fetal ovarian folliculogenesis

We recently demonstrated that the number of primordial follicles was significantly reduced in the ovaries of near-term baboon fetuses deprived of estrogen in utero and restored to normal in animals administered estradiol. Although the baboon fetal ovary expressed estrogen receptors alpha and beta, the mechanism(s) of estrogen action remains to be determined. It is well established that inhibin and activins function as autocrine/paracrine factors that impact adult ovarian function. However, our understanding of the expression of these factors in the primate fetal ovary is incomplete. Therefore, we determined the expression of alpha-inhibin, activin beta(A), activin beta(B), and activin receptors in fetal ovaries obtained at mid and late gestation from untreated baboons and at late gestation from animals in which fetal estrogen levels were reduced by >95% by maternal administration of the aromatase inhibitor CGS 20267 or restored to 30% of normal by treatment with CGS 20267 and estradiol benzoate. Immunocytochemical expression of alpha-inhibin was minimal to nondetectable in fetal ovaries from untreated baboons. In contrast, in baboons depleted of estrogen, alpha-inhibin was abundantly expressed in pregranulosa cells of interfollicular nests and granulosa cells of primordial follicles. Thus, the number (mean +/- SEM) per 0.08 mm2 of fetal ovarian cells expressing alpha-inhibin, determined by image analysis, was similar at mid and late gestation and increased approximately 8-fold (P < 0.01) near term in baboons treated with CGS 20267 and was restored (P < 0.01) to normal in baboons treated with CGS 20267 plus estradiol. Activin beta(A) was detected in oocytes and pregranulosa cells at midgestation and in oocytes and granulosa cells of primordial follicles at late gestation. Activin beta(B) was also expressed in pregranulosa cells and granulosa cells at mid and late gestation, respectively, but was not detected in oocytes. Neither the pattern nor the apparent level of expression of activin beta(A) or beta(B) were altered in fetal ovaries of baboons treated with CGS 20267 or CGS 20267 and estrogen. Activin receptors IA, IB, IIA, and IIB were detected by Western blot analysis in fetal ovaries at mid and late gestation, and expression was not altered by treatment with CGS 20267 or CGS 20267 and estrogen. Activin receptors IB and IIA were localized to oocytes and pregranulosa cells at midgestation and to granulosa cells and oocytes of primordial follicles at late gestation. Thus, the decrease in the number of follicles in the primate fetal ovary of baboons deprived of estrogen in utero was associated with increased expression of alpha-inhibin. Therefore, we propose that estrogen regulates fetal ovarian follicular development by controlling alpha-inhibin expression and, thus, the intraovarian inhibin:activin ratio.     

Role of nitric oxide in liver injury

The complex role of nitric oxide (NO) in the liver can be explained by its patterns of regulation and unique biochemical properties. With a broad range of direct and indirect molecular targets, NO acts as an inhibitor or agonist of cell signaling events. In the liver, constitutively generated NO maintains the hepatic microcirculation and endothelial integrity, while inducible NO synthase (iNOS)-governed NO production can be either beneficial or detrimental. For instance, NO potentiates the hepatic oxidative injury in warm ischemia/reperfusion, while iNOS expression protects against hepatic apoptotic cell death seen in models of sepsis and hepatitis. Anti-apoptotic actions are either cyclic nucleotide dependent or independent, including the expression of heat shock proteins, prevention of mitochondrial dysfunction, and inhibition of caspase activity by S-nitrosation. Whether NO protects or injures is probably determined by the type of insult, the abundance of reactive oxygen species (ROS), the source and amount of NO production and the cellular redox status of liver. Through the use of pharmacological NO donors or NOS gene transfer in conjunction with genetically altered knockout animals, the physiological and pathophysiological roles of NO in liver function can be explored in more detail. The purpose of this paper is to review the current understanding of the role of NO in liver injury.     

Signaling and function of caspase and c-jun N-terminal kinase in cisplatin-induced apoptosis

Caspases and c-Jun N-terminal kinase (JNK) are activated in tumor cells during induction of apoptosis. We investigated the signaling cascade and function of these enzymes in cisplatin-induced apoptosis. Treatment of Jurkat T-cells with cisplatin induced cell death with DNA fragmentation and activation of caspase and JNK. Bcl-2 overexpression suppressed activation of both enzymes, whereas p35 and CrmA inhibited only the DEVDase (caspase-3-like) activity, indicating that the activation of these enzymes may be differentially regulated. Cisplatin induced apoptosis with the cytochrome c release and caspase-3 activation in both wild-type and caspase-8-deficient JB-6 cells, while the Fas antibody induced these apoptotic events only in wild-type cells. This indicates that caspase-8 activation is required for Fas-mediated apoptosis, but not cisplatin-induced cell death. On the other hand, cisplatin induced the JNK activation in both the wild-type and JB-6 cells, and the caspase-3 inhibitor Z-DEVD-fmk did not inhibit this activation. The JNK overexpression resulted in a higher JNK activity, AP-1 DNA binding activity, and metallothionein expression than the empty vector-transfected cells following cisplatin treatment. It also partially protected the cells from cisplatin-induced apoptosis by decreasing DEVDase activity. These data suggest that the cisplatin-induced apoptotic signal is initiated by the caspase-8-independent cytochrome c release, and the JNK activation protects cells from cisplatin-induced apoptosis via the metallothionein expression.