Hepatic ischemia/reperfusion injury is an important complication of liver surgery and transplantation. The mechanisms of this injury as well as the subsequent reparative and regenerative processes have been the subject of thorough study. In this paper, we discuss the complex and coordinated responses leading to parenchymal damage after liver ischemia/reperfusion as well as the manner in which the liver clears damaged cells and regenerates functional mass.
AIM: To investigate the mechanisms of liver growth and atrophy after portal vein ligation (PVL) and its effects on tumor growth.
METHODS: Mice were subjected to PVL, partial hepatectomy, or sham surgery. The morphological alterations, activation of transcription factors, and expression of cytokines and growth factors involved in liver regeneration were evaluated. In a separate set of experiments, murine colorectal carcinoma cells were injected via the portal vein and the effect of each operation on liver tumor growth was studied.
RESULTS: Liver regeneration after PVL and partial hepatectomy were very similar. In ligated lobes, various cytokines, transcription factors and regulatory factors were significantly upregulated compared to non-ligated lobes after PVL. Atrophy in ligated lobes was a result of early necrosis followed by later apoptosis. Tumor growth was significantly accelerated in ligated compared to non-ligated lobes.
CONCLUSION: Tumor growth was accelerated in ligated liver lobes and appeared to be a result of increased growth factor expression.
Portal vein ligation; Tumor growth; Growth factor; Atrophy; Apoptosis
Traumatic brain injury (TBI) is a leading cause of mortality and disability. Acute postinjury insults after TBI, such as hypoxia, contribute to secondary brain injury and worse clinical outcomes. The functional and neuroinflammatory effects of brief episodes of hypoxia experienced following TBI have not been evaluated. Our previous studies have identified interleukin 6 (IL-6) as a potential mediator of mild TBI–induced pathology. In the present study, we sought to determine the effects of brief hypoxia on mild TBI and whether IL-6 played a role in the neuroinflammatory and functional deficits after injury. A murine model of mild TBI was induced by a weight drop (500 g from 1.5 cm). After injury, mice were exposed to immediate hypoxia (Fio2 = 15.1%) or normoxia (Fio2 = 21%) for 30 min. Serum and brain samples were analyzed for inflammatory cytokines 24 h after TBI. Neuron-specific enolase was measured as a serum biomarker of brain injury. Evaluation of motor coordination was performed for 5 days after TBI using a rotarod device. In some animals, anti–IL-6 was administered following TBI and hypoxia to neutralize systemic IL-6. Mice undergoing TBI had significant increases in brain injury. Exposure to brief hypoxia after TBI resulted in a more than 5-fold increase in serum neuron-specific enolase. This increase was associated with increases in serum and brain cytokine expression, suggesting that brief hypoxia exacerbates systemic and brain inflammation. Neutralization of IL-6 suppressed postinjury neuroinflammation and neuronal injury. In addition, TBI and hypoxia induced significant motor coordination deficits that were completely abrogated by IL-6 blockade. Exposure to hypoxia after TBI induces neuroinflammation and brain injury. These changes can be mitigated by neutralization of systemic IL-6. Interleukin 6 blockade also corrected the TBI-induced deficit in motor coordination. These data suggest that systemic IL-6 modulates the degree of neuroinflammation and contributes to reduced motor coordination after mild TBI.
Traumatic brain injury; neuroinflammation; motor coordination; hypoxia; trauma; inflammation
Previous studies have demonstrated the significance of signaling through the CXCR2 receptor in the process of recovery and regeneration of functional liver mass after hepatic ischemia/reperfusion (I/R). CXCR2 is constitutively expressed on both neutrophils and hepatocytes, however, the cell-specific roles of this receptor is unknown. In the present study, chimeric mice were created through bone marrow transplantation (BMT) using wild-type and CXCR2-knockout mice, yielding selective expression of CXCR2 on hepatocytes (Hep) and/or myeloid cells (My) in the following combinations: Hep+/My+; Hep−/My+; Hep+/My−; Hep−/My−. These tools allowed us to assess the contributions of myeloid and hepatocyte CXCR2 in the recovery of the liver after I/R injury. Flow cytometry confirmed adoption of the donor phenotype in neutrophils. Interestingly, Kupffer cells from all chimeras lacked CXCR2 expression. Recovery/regeneration of hepatic parenchyma was assessed by histologic assessment and measurement of hepatocyte proliferation. CXCR2Hep+/My+ mice showed the least amount of liver recovery and hepatocyte proliferation, while CXCR2Hep−/My− mice had the greatest liver recovery and hepatocyte proliferation. CXCR2Hep+/My− mice had enhanced liver recovery with hepatocyte proliferation similar to CXCR2Hep−/My− mice. Myeloid expression of CXCR2 directly regulated CXC chemokine expression levels following hepatic I/R, such that mice lacking myeloid CXCR2 had markedly increased chemokine expression compared to mice expressing CXCR2 on myeloid cells.
The data suggest that CXCR2 on myeloid cells is the predominant regulator of liver recovery and regeneration after I/R injury, while hepatocyte CXCR2 plays a minor, secondary role. These findings suggest that myeloid cell-directed therapy may significantly impact liver regeneration after liver resection or transplantation.
chemokines; neutrophils; hepatocytes; hepatic regeneration; bone marrow transplant
Background and Aim
The purpose of the present study was to determine the effects of IL-37 on liver cells and on liver inflammation induced by hepatic ischemia/reperfusion (I/R).
Materials and methods
Mice were subjected to I/R. Some mice received recombinant IL-37 (IL-37) at the time of reperfusion. Serum levels of alanine amino transferase, liver myeloperoxidase content were assessed. Serum and liver TNFα, MIP-2 and KC were also assessed. Hepatic reactive oxygen species (ROS) levels were assessed. For in vitro experiments, isolated hepatocytes and Kupffer cells were treated with IL-37 and inflammatory stimulants. Cytokine and chemokine production by these cells were assessed. Primary hepatocytes were induced cell injury and treated with IL-37 concurrently. Hepatocyte cytotoxicity and Bcl-2 expression were determined. Isolated neutrophils were treated with TNFα and IL-37 and neurtrophil activation and respiratory burst were assessed.
IL-37 reduced hepatocyte injury and neutrophil accumulation in the liver after I/R. These effects were accompanied by reduced serum levels of TNFα and MIP-2 and hepatic ROS levels.IL-37 significantly reduced MIP-2 and KC productions from LPS-stimulated hepatocytes and Kupffer cells. IL-37 significantly reduced cell death and increased Bcl-2 expression in hepatocytes. IL-37 significantly suppressed TNFα induced-neutrophil activation.
IL-37 is protective against hepatic I/R injury. These effects are related to the ability of IL-37 to reduce proinflammatory cytokine and chemokine production by hepatocytes and Kupffer cells as well as having a direct protective effect on hepatocytes. In addition, IL-37 contributes to reduce liver injury through suppression of neutrophil activity.
liver ischemia/reperfusion; inflammation; chemokines
IL-33 is a recently identified member of the IL-1 family that binds to the receptor, ST2L. In the current study, we sought to determine if IL-33 is an important in the hepatic response to I/R. Male C57BL/6 mice were subjected to 90 minutes of partial hepatic ischemia followed by up to 8 hours of reperfusion. Some mice received recombinant IL-33 (IL-33) intraperitoneally prior to surgery or anti-ST2 antibody intraperitoneally at the time of reperfusion. Primary hepatocytes and Kupffer cells were isolated and treated with IL-33 to assess the effects of IL-33 on inflammatory cytokine production. Primary hepatocytes were treated with IL-33 to assess the effects of IL-33 on the mediators for cell survival in hepatocytes. IL-33 protein expression increased within 4 hours after reperfusion and remained elevated for up to 8 hours. ST2L protein expression was detected in normal liver and was upregulated within 1hr and peaked at 4hrs after I/R. ST2L was primarily expressed by hepatocytes, with little to no expression by Kupffer cells. IL-33 significantly reduced hepatocellular injury and liver neutrophil accumulation at 1 hour and 8 hours after reperfusion. In addition, IL-33 treatment increased liver activation of NF-κB, p38 mitogen activated protein kinase (MAPK), Cyclin D1 and Bcl-2, but reduced serum levels of CXC chemokines. In vitro experiments demonstrated that IL-33 significantly reduced hepatocyte cell death, due to increased NF-κB activation and Bcl-2 expression in hepatocytes. Conclusion: The data suggest that IL-33 is an important endogenous regulator of hepatic I/R injury. It appears that IL-33 has direct protective effects on hepatocytes, associated with activation of NF-κB, p38 MAPK, Cyclin D1 and Bcl-2 that limits liver injury and reduces the stimulus for inflammation.
liver injury; hepatocytes; inflammation; cytokines; chemokines
The transcription factor NF-κB plays diverse roles in the acute injury response to hepatic ischemia/reperfusion (I/R). Activation of NF-κB in Kupffer cells promotes inflammation through cytokine expression while activation in hepatocytes may be cell protective. The interaction of receptor activator of NF-κB (RANK) and its ligand (RANKL) promotes NF-κB activation, however this ligand-receptor system has not been studied in acute liver injury. In the current study, we sought to determine if RANK and RANKL were important in the hepatic response to I/R. Mice were subjected to partial hepatic ischemia followed by reperfusion. In some experiments, mice received recombinant RANKL or neutralizing antibodies to RANKL 1h prior to surgery or at reperfusion to assess the role of RANKL/RANK signaling during I/R injury. RANK was constitutively expressed in the liver and was not altered by I/R. RANK was strongly expressed in hepatocytes and very weakly expressed in Kupffer cells. Serum RANKL concentrations increased after I/R and peaked 4 hours after reperfusion. Serum levels of osteoprotegerin (OPG), a decoy receptor for RANKL, steadily increased over the 8 hour period of reperfusion. Treatment with RANKL, before ischemia or at reperfusion, increased hepatocyte NF-κB activation and significantly reduced liver injury. These beneficial effects occurred without any effect on cytokine expression or liver inflammation. Treatment with anti-RANKL antibodies had no effect on liver I/R injury.
During the course of injury, endogenous OPG appears to suppress the effects of RANKL. However, exogenous administration of RANKL, given either prophylactically or post-injury, reduces liver injury in a manner associated with increased hepatocyte NF-κB activation. The data suggest that RANK may be a viable therapeutic target in acute liver injury.
liver injury; hepatocytes; osteoprotegerin; inflammation; cytokines
Hepatic ischemia/reperfusion (I/R) injury is an important complication of liver surgery and transplantation. Regulation of this injury response occurs at the cellular and molecular levels. Previous studies have shown that interleukin-6 (IL-6) is a negative regulator of the acute inflammatory injury occurring as a result of hepatic I/R. The Signal Transducer and Activator of Transcription-3 (STAT3) is a key target of receptor signaling for IL-6. Both IL-6 and STAT3 have been implicated in the protective effects of ischemic preconditioning of the liver. However, there have been no studies that have directly addressed the potential role of STAT3 in regulating acute inflammatory liver injury induced by I/R. In the current study, we investigated whether blockade of STAT3 phosphorylation altered the injury response to hepatic I/R injury.
Male Balb/c mice were subjected to 90 minutes of partial hepatic ischemia followed by reperfusion with or without treatment with specific inhibitors of STAT3 activation, AG490 (selective JAK2 inhibitor) or STATTIC (direct inhibitor of STAT3 phosphorylation). Mice were sacrificed at 8 and 24 hours after reperfusion.
STAT3 activation was induced by I/R. This activation was partially inhibited by administration of AG490 and almost completely abrogated by treatment with STATTIC. Despite the blockade of STAT3, neither AG490 nor STATTIC had any effect on acute liver injury induced by I/R. Treatment with STATTIC did reduce hepatic neutrophil accumulation.
The data suggest that STAT3 is not a central regulator of acute liver injury induced by I/R.
STAT3; hepatic ischemia/reperfusion; neutrophil accumulation
The process of liver repair and regeneration following hepatic injury is complex and relies on a temporally coordinated integration of several key signaling pathways. Pathways activated by members of the CXC family of chemokines play important roles in the mechanisms of liver repair and regeneration through their effects on hepatocytes. However, little is known about the signaling pathways utilized by CXC chemokine receptors in hepatocytes. Here we review our current understanding of the pathways involved in both CXC chemokine receptor signaling in other cell types, most notably neutrophils, and similar pathways operant during hepatocyte proliferation/liver regeneration in order to formulate a basis for the function of CXC chemokine receptor signaling in hepatocytes.
Liver injury; ischemia/reperfusion; hepatectomy; CXCR1; CXCR2
CXC chemokines mediate hepatic inflammation and injury following ischemia/reperfusion (I/R). More recently, signaling through CXC chemokine receptor-2 (CXCR2) was shown to delay liver recovery and repair after I/R injury. The chemokine receptor, CXCR1 shares ligands with CXCR2, yet nothing is known about its potential role in liver pathology. In the present study, we examined the role of CXCR1 in the injury and recovery responses to I/R using a murine model. CXCR1 expression was undetectable in livers of sham-operated mice. However, after ischemia CXCR1 expression increased 24 hours of reperfusion and was maximal after 96 hours of reperfusion. CXCR1 expression was localized largely to hepatocytes. In order to assess the function of CXCR1, CXCR2-/- mice were treated with the CXCR1/CXCR2 antagonist, repertaxin. Prophylactic treatment with repertaxin had no effect on acute inflammation or liver injury. However, when repertaxin was administered 24 hours post-reperfusion there was a significant increase in hepatocellular injury and a delay in recovery compared to control-treated mice. CXCR1-/- mice also demonstrated delayed recovery and regeneration after I/R when compared to wild-type mice. In vitro, hepatocytes from CXCR2-/- mice that were stimulated to express CXCR1 showed increased proliferation in response to ligand. Hepatocyte proliferation was decreased in CXCR1-/- mice in vivo.
This is the first report to show that CXCR1 expression is induced in hepatocytes after injury. Furthermore, the data suggest that CXCR1 has divergent effects from CXCR2 and appears to facilitate repair and regenerative responses after I/R injury.
chemokines; chemokine receptors; liver repair; hepatocyte proliferation; regeneration
Fibrosis of the lung is one of the major clinical problems of cystic fibrosis and chronic obstructive pulmonary disease. However, the molecular mechanisms leading to pulmonary fibrosis are poorly characterized and require definition. Here, we demonstrate that chronic accumulation of ceramide in the lung contributes to the development of fibrosis in aged cystic fibrosis mice. Genetic or pharmacological normalization of ceramide in cystic fibrosis mice, which was achieved by heterozygosity of acid sphingomyelinase or chronic (6.5 month long) treatment of mice with pharmacological inhibitors of acid sphingomyelinase significantly decreased the development of lung fibrosis. Moreover, our studies demonstrate that long-term treatment of cystic fibrosis mice with pharmacological inhibitors of acid sphingomyelinase or genetic heterozygosity of the enzyme also minimizes pulmonary inflammatory cytokines in cystic fibrosis mice. This data identifies ceramide as a key molecule associated with pulmonary fibrosis in cystic fibrosis mice and demonstrate for the first time that prolonged inhibition of acid sphingomyelinase is able to attenuate fibrosis and inflammation in this animal model.
Ceramide; Cystic fibrosis; Acid sphingomyelinase; Cftr
Our previous work suggested an important role for the peptidyl-prolyl isomerase, Pin1, in hepatic NF-κB activation and liver injury during ischemia/reperfusion (I/R). In this study, we sought to determine the function of Pin1 in the injury response to hepatic I/R.
Wild-type and Pin1-/- mice were subjected to partial hepatic I/R. In addition, hepatocytes and Kupffer cells were isolated from these mice.
Pin1-/- mice had reduced hepatic NF-κB activation and more liver injury after I/R than wild-type mice. The increased injury was not a result of enhanced inflammation as Pin1-/- mice had the same level of proinflammatory cytokine production and less neutrophil accumulation in the liver. The reduced NF-κB activation was not a result of a defect in nuclear translocation of NF-κB. In fact, hepatic nuclear p65 protein expression was higher in Pin1-/- mice than wild-type mice. This suggests that Pin1 is important for NF-κB-DNA binding. This effect was specific to hepatocytes as isolated Kupffer cells from wild-type and Pin1-/- mice were identical in their activation of NF-κB and production of cytokines after stimulation. In contrast, hepatocytes stimulated with TNFα had greatly reduced NF-κB activation, reduced production of the CXC chemokine, MIP-2, and increased cell death.
These data suggest that Pin1 is a critical regulator of NF-κB activation in hepatocytes and its role in these cells appears to confer direct protective effects.
liver; hepatocytes; ischemia/reperfusion; Pin1; NF-κB
Hepatic ischemia/reperfusion (I/R) leads to liver injury and dysfunction through the initiation of a biphasic inflammatory response that is regulated by the transcription factor, NF-κB. We have previously shown that there is an age-dependent difference in the injury response to hepatic I/R in mice that correlates with divergent activation of NF-κB such that young mice have greater NF-κB activation, but less injury than old mice. In the present study, we investigated the mechanism by which age alters the activation of NF-κB in the liver during I/R. Young (4-5 weeks) and old (12-14 months) mice underwent partial hepatic ischemia/reperfusion. Livers were obtained for RNA microarray analysis and protein expression assays. Using microarray analysis, we identified age-dependent differences in the expression of genes related to protein ubiquitinylation and the proteasome. In old mice, genes that are involved in the ubiquitin-proteasome pathway were significantly down-regulated during I/R. Consistent with these findings, expression of a critical proteasome subunit, non-ATPase 4 (PSMD4), was reduced in old mice. Expression of the NF-κB-inhibitory protein, IκBα, was increased in old mice and was greatly phosphorylated and ubiquitinylated. The data provide strong evidence that the age-related defect in hepatic NF-κB signaling during I/R is a result of decreased expression of PSMD4, a proteasome subunit responsible for recognition and recruitment of ubiquitinylated substrates to the proteasome. It appears that decreased PSMD4 expression prevents recruitment of phosphorylated and ubiquitinylated IκBα to the proteasome, resulting in a defect in NF-κB activation.
liver injury; inflammation; ageing; genomics; microarray
Immune-complexes play an important role in the inflammatory diseases of the lung. Neutrophil activation mediates immune-complex (IC) deposition-induced acute lung injury (ALI). Components of gamma amino butyric acid (GABA) signaling, including GABA B receptor 2 (GABABR2), GAD65/67 and the GABA transporter, are present in the lungs and in the neutrophils. However, the role of pulmonary GABABR activation in the context of neutrophil-mediated ALI has not been determined. Thus, the objective of the current study was to determine whether administration of a GABABR agonist, baclofen would ameliorate or exacerbate ALI. We hypothesized that baclofen would regulate IC-induced ALI by preserving pulmonary GABABR expression. Rats were subjected to sham injury or IC-induced ALI and two hours later rats were treated intratracheally with saline or 1 mg/kg baclofen for 2 additional hours and sacrificed. ALI was assessed by vascular leakage, histology, TUNEL, and lung caspase-3 cleavage. ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF). Moreover, ALI decreased lung GABABR2 expression, increased phospho-p38 MAPK, promoted IκB degradation and increased neutrophil influx in the lung. Administration of baclofen, after initiation of ALI, restored GABABR expression, which was inhibited in the presence of a GABABR antagonist, CGP52432. Baclofen administration activated pulmonary phospho-ERK and inhibited p38 MAPK phosphorylation and IκB degradation. Additionally, baclofen significantly inhibited pro-inflammatory TNF-α and IL-1βAcP release and promoted BAL neutrophil apoptosis. Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling. Interestingly, GABABR2 expression was regulated in the type II pneumocytes in lung tissue sections from lung injured patients, further suggesting a physiological role for GABABR2 in the repair process of lung damage. GABABR2 agonists may play a potential therapeutic role in ALI.
CXC chemokines and their receptor, CXCR2, are important components of the hepatic inflammatory response to ischemia/reperfusion. However, direct effects of CXC chemokines on hepatocytes during this response have not been studied. Wild-type and CXCR2-/- mice were subjected to 90 minutes of partial hepatic ischemia followed by up to 96 hours of reperfusion. CXCR2-/- mice had significantly less liver injury at all reperfusion times compared to wild-type mice. Early neutrophil recruitment (12 hours) was diminished in CXCR2-/- mice, but within 24 hours was the same as wild-type mice. Hepatocyte proliferation and regeneration was accelerated in CXCR2-/- mice compared to wild-type mice. These effects were associated with increased activation of NF-κB and STAT3, despite there being no difference in the expression of proliferative factors such as TNFα, IL-6, and HGF. To establish whether the accelerated proliferation and regeneration observed in CXCR2-/- mice was due to effects on hepatocytes rather than just a generalized decrease in acute inflammatory injury, mice were treated with the CXCR2 antagonist, SB225002, after neutrophil recruitment and injury were maximal (24 hours after reperfusion). SB225002 treatment increased hepatocyte proliferation and regeneration in a manner identical to that observed in CXCR2-/- mice. Treatment of primary wild-type hepatocytes with MIP-2 showed that low concentrations protected against cell death whereas high concentrations induced cell death. These effects were absent in hepatocytes from CXCR2-/- mice.
The data suggest that hepatocyte CXCR2 regulates proliferation and regeneration after I/R injury and reveal important differences in the role of this receptor in liver regeneration and repair induced under different conditions that may be related to ligand concentration.
liver injury; chemokines; regeneration; inflammation; proliferation
Hepatic ischemia/reperfusion (I/R) injury is a complication of liver surgery, transplantation and shock and is known to be age-dependent. Our laboratory has recently shown that peroxisome proliferator-activated receptor-gamma (PPARγ) is downregulated during hepatic ischemia and that this exacerbates injury. Here we examined whether activation of PPARγ during ischemia was age-dependent. Male mice of different ages (young: 4–5 weeks; adult: 10–12 weeks; old: 10–12 months) were subjected to up to 90 minutes of hepatic ischemia. PPARγ activation occurred throughout ischemia in young mice, whereas activation in adult and old mice was lost after 30 minutes. No significant differences were noted in PPARγ ligand expression amongst the age groups. However, in young mice we observed a predominance of PPARγ1 in the nucleus, whereas in old mice this isoform remained largely in the cytoplasm. Finally, the degree of PPARγ activation was associated with autophagy in the liver, a mechanism of self-preservation.
PPARγ activation is prolonged in young mice as compared to older mice. This appears to be mediated by a selective retention of PPARγ1 in the nucleus and is associated with increased autophagy. The data suggest that PPARγ activation is an important component of the age-dependent response to hepatic I/R injury.
liver injury; transcription; autophagy; hepatocytes
Our previous in vitro studies have demonstrated dose-dependent effects of CXCR2 ligands on hepatocyte cell death and proliferation. In the current study, we sought to determine if CXCR2 ligand concentration is responsible for the divergent effects of these mediators on liver regeneration after ischemia/reperfusion injury and partial hepatectomy.
Murine models of partial ischemia/reperfusion injury and hepatectomy were used to study the effect of CXCR2 ligands on liver regeneration.
We found that hepatic expression of the CXCR2 ligands, macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), was significantly increased after both I/R injury and partial hepatectomy. However, expression of these ligands after I/R injury was 30-100-fold greater than after hepatectomy. Interestingly, the same pattern of expression was found in ischemic versus non-ischemic liver lobes following I/R injury with expression significantly greater in the ischemic liver lobes. In both systems, lower ligand expression was associated with increased hepatocyte proliferation and liver regeneration in a CXCR2-dependent fashion. To confirm that these effects were related to ligand concentration, we administered exogenous MIP-2 and KC to mice undergoing partial hepatectomy. Mice received a “high” dose that replicated serum levels found after I/R injury and a “low” dose that was similar to that found after hepatectomy. Mice receiving the “high” dose had reduced levels of hepatocyte proliferation and regeneration whereas the “low” dose promoted hepatocyte proliferation and regeneration.
Together, these data demonstrate that concentrations of CXC chemokines regulate the hepatic proliferative response and subsequent liver regeneration.
Background & Aims
Hepatic stellate cells (HSCs) that express glial fibrillary acidic protein (GFAP) are located between the sinusoidal endothelial cells and hepatocytes. HSCs are activated during liver injury and cause hepatic fibrosis by producing excessive extracellular matrix. HSCs also produce many growth factors, chemokines and cytokines, and thus may play an important role in acute liver injury. However, this function has not been clarified due to unavailability of a model in which HSCs are depleted from the normal liver.
We treated mice expressing HSV-thymidine kinase under the GFAP promoter (GFAP-Tg) with 3 consecutive (3 days apart) CCl4 (0.16 μl/g; ip) injections to stimulate HSCs to enter the cell cycle and proliferate. This was followed by 10-day ganciclovir (40 μg/g/day; ip) treatment, which is expected to eliminate actively proliferating HSCs. Mice were then subjected to hepatic ischemia/reperfusion (I/R) or endotoxin treatment.
CCl4/ganciclovir treatment caused depletion of the majority of HSCs (about 64-72%), while the liver recovered from the initial CCl4-induced injury (confirmed by histology, serum ALT and neutrophil infiltration). The magnitude of hepatic injury due to I/R or endotoxemia (determined by histopathology and serum ALT) was lower in HSC-depleted mice. Their hepatic expression of TNF-α, neutrophil chemoattractant CXCL1 and endothelin-A receptor also was significantly lower than the control mice.
HSCs play an important role both in I/R- and endotoxin-induced acute hepatocyte injury, with TNF-α and endothelin-1 as important mediators of these effects.
Hepatic stellate cells; Depletion; Ischemia/Reperfusion; Endotoxemia; Lipopolysaccharide; Hepatocytes; Liver; Injury; Necrosis; Apoptosis
During chronic caloric excess, adipose tissue expands primarily by enlargement of individual adipocytes, which become stressed with lipid overloading, thereby contributing to obesity-related disease. Although adipose tissue contains numerous preadipocytes, differentiation into functionally competent adipocytes is insufficient to accommodate the chronic caloric excess and prevent adipocyte overloading. We report for the first time that a chronic high-fat diet (HFD) impairs adipogenic differentiation, leading to accumulation of inefficiently differentiated adipocytes with blunted expression of adipogenic differentiation-specific genes. Preadipocytes from these mice likewise exhibit impaired adipogenic differentiation, and this phenotype persists during in vitro cell culture. HFD-induced impaired adipogenic differentiation is associated with elevated expression of histone deacetylase 9 (HDAC9), an endogenous negative regulator of adipogenic differentiation. Genetic ablation of HDAC9 improves adipogenic differentiation and systemic metabolic state during an HFD, resulting in diminished weight gain, improved glucose tolerance and insulin sensitivity, and reduced hepatosteatosis. Moreover, compared with wild-type mice, HDAC9 knockout mice exhibit upregulated expression of beige adipocyte marker genes, particularly during an HFD, in association with increased energy expenditure and adaptive thermogenesis. These results suggest that targeting HDAC9 may be an effective strategy for combating obesity-related metabolic disease.
Resuscitation of patients after hemorrhage often results in pulmonary inflammation and places them at risk for the development of acute respiratory distress syndrome. Our previous data indicate that macrophage-derived chemokine (MDC/CCL22) is elevated after resuscitation, but its direct role in this inflammatory response is unknown. MDC signaling through the C-C chemokine receptor type 4 (CCR4) is implicated in other pulmonary proinflammatory conditions, leading us to hypothesize that MDC may also play a role in the pathogenesis of lung inflammation following hemorrhage and resuscitation. To test this, C57BL/6 mice underwent pressure-controlled hemorrhage followed by resuscitation with lactated Ringer’s solution. Pulmonary inflammation and inflammatory cell recruitment were analyzed with histological staining, and serum- and tissue-level cytokines were measured by ELISA. Pulmonary inflammation and cell recruitment following hemorrhage and resuscitation were associated with systemic MDC levels. Inhibition of MDC via injection of a specific neutralizing antibody prior to hemorrhage and resuscitation significantly reduced pulmonary levels of the chemotactic cytokines KC, MIP-2, and MIP-1α as well as inflammatory cell recruitment to the lungs. Intravenous administration of recombinant MDC prior to resuscitation augmented pulmonary inflammation and cell recruitment. Histological evaluation revealed the expression of CCR4 within the bronchial epithelium, and in vitro treatment of activated bronchial epithelial cells with MDC resulted in production and secretion of neutrophil chemokines. The present study identifies MDC as a novel mediator of lung inflammation after hemorrhage and resuscitation. MDC neutralization may provide a therapeutic strategy to mitigate this inflammatory response.
hemorrhagic shock; pulmonary neutrophil recruitment; CCR4; bronchial epithelium
Traumatic brain injury (TBI) initiates a neuroinflammatory response that increases the risk of TBI-related mortality. Acute alcohol intoxication at the time of TBI is associated with improved survival. Ethanol is recognized as a systemic immunomodulator that may also impart neuroprotection. The effects of alcohol on TBI-induced neuroinflammation, however, are unknown. We hypothesized that ethanol treatment prior to TBI may provide neuroprotection by diminishing the neuroinflammatory response to injury.
Materials and methods
Mice underwent gavage with ethanol (EtOH) or water (H2O) prior to TBI. Animals were subjected to blunt TBI or sham injury (Sham). Posttraumatic rapid righting reflex (RRR) and apnea times were assessed. Cerebral and serum samples were analyzed by ELISA for inflammatory cytokine levels. Serum neuron-specific enolase (NSE), a biomarker of injury severity, was also measured.
Neurologic recovery from TBI was more rapid in H2O-treated mice compared with EtOH-treated mice. However, EtOH/TBI mice had a 4-fold increase in RRR time compared with EtOH/Sham, whereas H2O/TBI mice had a 15-fold increase in RRR time compared with H2O/Sham. Ethanol intoxication at the time of TBI significantly increased posttraumatic apnea time. Preinjury EtOH treatment was associated with reduced levels of proinflammatory cytokines IL-6, KC, MCP-1, and MIP-1α post TBI. NSE was significantly increased post injury in the H2O/TBI group compared with H2O/Sham but was not significantly reduced by EtOH pretreatment.
Alcohol treatment prior to TBI reduces the local neuroinflammatory response to injury. The decreased neurologic and inflammatory impact of TBI in acutely intoxicated patients may be responsible for improved clinical outcomes.
Traumatic brain injury; Alcohol; Inflammation; Neuroinflammation
Traumatic brain injury results in significant morbidity and mortality and is associated with infectious complications, particularly pneumonia. However, whether traumatic brain injury directly impacts the host response to pneumonia is unknown. The objective of this study was to determine the nature of the relationship between traumatic brain injury and the prevalence of pneumonia in trauma patients and investigate the mechanism of this relationship using a murine model of traumatic brain injury with pneumonia.
Data from the National Trauma Data Bank and a murine model of traumatic brain injury with postinjury pneumonia.
Academic medical centers in Cincinnati, OH, and Boston, MA.
Trauma patients in the National Trauma Data Bank with a hospital length of stay greater than 2 days, age of at least 18 years at admission, and a blunt mechanism of injury. Subjects were female ICR mice 8–10 weeks old.
Administration of a substance P receptor antagonist in mice.
Measurements and Main Results
Pneumonia rates were measured in trauma patients before and after risk adjustment using propensity scoring. In addition, survival and pulmonary inflammation were measured in mice undergoing traumatic brain injury with or without pneumonia. After risk adjustment, we found that traumatic brain injury patients had significantly lower rates of pneumonia compared to blunt trauma patients without traumatic brain injury. A murine model of traumatic brain injury reproduced these clinical findings with mice subjected to traumatic brain injury demonstrating increased bacterial clearance and survival after induction of pneumonia. To determine the mechanisms responsible for this improvement, the substance P receptor was blocked in mice after traumatic brain injury. This treatment abrogated the traumatic brain injury–associated increases in bacterial clearance and survival.
The data demonstrate that patients with traumatic brain injury have lower rates of pneumonia compared to non–head-injured trauma patients and suggest that the mechanism of this effect occurs through traumatic brain injury–induced release of substance P, which improves innate immunity to decrease pneumonia.
infection; innate immunity; leukocytes; lung inflammation; neuropeptide; vagus nerve
Members of the signal transducer and activator of transcription (STAT) family are transcription factors that mediate many of the effects of pro- and anti-inflammatory cytokines. The progressive systemic inflammatory response induced by endotoxin is mediated by overzealous cytokine production. Here we identify STAT4 and STAT6 as critical regulators of the systemic inflammatory response to endotoxin. Mice deficient for STAT4 or STAT6 were highly susceptible to lethal endotoxemia. In STAT4–/– mice, antibody blockade of IL-12 prevented mortality, suggesting that STAT4 confers protection, while another signaling pathway mediates the detrimental effects of IL-12. In STAT6–/– mice we observed dysregulated activation of the transcription factor NF-κB, resulting in augmented production of proinflammatory cytokines and chemokines. Furthermore, STAT6–/– mice displayed increased organ accumulation of leukocytes and significant hepatocellular injury. These findings demonstrate that STAT4 and STAT6 confer protection against endotoxin-induced death and that for STAT6 these protective effects occur through the regulation of NF-κB activation and subsequent production of proinflammatory cytokines and chemokines.
Mild traumatic brain injury is a serious public health concern, affecting more than 1.7 million people in the United States annually. Mild TBI is difficult to diagnose and is clinically associated with impaired motor coordination and cognition.
Mice were subjected to a mild TBI (mTBI-1 or mTBI-2) induced by a weight drop model. Brain injury was assessed histologically and biochemically, the latter by serum neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP). Systemic and brain inflammation were measured by cytokine array. Blood brain barrier (BBB) integrity was determined by cerebral vascular leakage of micromolecular and macromolecular fluorescent molecules. Mice were evaluated using a rotarod device and novel object recognition to measure motor coordination and cognition, respectively.
Mice undergoing mTBI-1 or mTBI-2 had significant deficits in motor coordination and cognition for several days after injury compared to controls. Furthermore, we found both mTBI-1 and mTBI-2 caused micromolecular leakage in the BBB, whereas only mTBI-2 caused macromolecular leakage. Serum NSE and GFAP were elevated acutely and corresponded to the degree of injury, but returned to baseline within 24 hours. Serum cytokines interleukin-6 (IL-6) and keratinocyte-derived chemokine (KC) were significantly increased within 90 minutes of TBI. IL-6 levels correlated with the degree of injury.
The current study provides a reproducible model of mild TBI in mice that exhibits pathological features of mild TBI in humans. Furthermore, our data suggest that serum cytokines, such as IL-6, may be effective biomarkers for severity of head injury.
TBI; inflammation; combat casualty care; trauma; cytokines; chemokines
Cystic fibrosis patients and patients with chronic obstructive pulmonary disease, trauma, burn wound, or patients requiring ventilation are susceptible to severe pulmonary infection by Pseudomonas aeruginosa. Physiological innate defense mechanisms against this pathogen, and their alterations in lung diseases, are for the most part unknown. We now demonstrate a role for the sphingoid long chain base, sphingosine, in determining susceptibility to lung infection by P. aeruginosa. Tracheal and bronchial sphingosine levels were significantly reduced in tissues from cystic fibrosis patients and from cystic fibrosis mouse models due to reduced activity of acid ceramidase, which generates sphingosine from ceramide. Inhalation of mice with sphingosine, with a sphingosine analog, FTY720, or with acid ceramidase rescued susceptible mice from infection. Our data suggest that luminal sphingosine in tracheal and bronchial epithelial cells prevents pulmonary P. aeruginosa infection in normal individuals, paving the way for novel therapeutic paradigms based on inhalation of acid ceramidase or of sphingoid long chain bases in lung infection.
cystic fibrosis; long chain base; lung infection; Pseudomonas aeruginosa; sphingosine