An alteration of the gut-liver axis drives pulmonary inflammation after intoxication and burn injury in mice.

Approximately half of all adult burn patients are intoxicated at the time of their injury and have worse clinical outcomes than those without prior alcohol exposure. This study tested the hypothesis that intoxication alters the gut-liver axis, leading to increased pulmonary inflammation mediated by burn-induced IL-6 in the liver. C57BL/6 mice were given 1.2 g/kg ethanol 30 min prior to a 15% total body surface area burn. To restore gut barrier function, the specific myosin light chain kinase inhibitor membrane-permeant inhibitor of kinase (PIK), which we have demonstrated to reduce bacterial translocation from the gut, was administered 30 min after injury. Limiting bacterial translocation with PIK attenuated hepatic damage as measured by a 47% reduction in serum alanine aminotransferase (P < 0.05), as well as a 33% reduction in hepatic IL-6 mRNA expression (P < 0.05), compared with intoxicated and burn-injured mice without PIK. This mitigation of hepatic damage was associated with a 49% decline in pulmonary neutrophil infiltration (P < 0.05) and decreased alveolar wall thickening compared with matched controls. These results were reproduced by prophylactic reduction of the bacterial load in the intestines with oral antibiotics before intoxication and burn injury. Overall, these data suggest that the gut-liver axis is deranged when intoxication precedes burn injury and that limiting bacterial translocation in this setting attenuates hepatic damage and pulmonary inflammation.

Enteropathogenic E. coli effectors EspG1/G2 disrupt microtubules, contribute to tight junction perturbation and inhibit restoration.

Enteropathogenic Escherichia coli (EPEC) uses a type 3 secretion system to transfer effector proteins into the host intestinal epithelial cell. Several effector molecules contribute to tight junction disruption including EspG1 and its homologue EspG2 via a mechanism thought to involve microtubule destruction. The aim of this study was to investigate the contribution of EspG-mediated microtubule disruption to TJ perturbation. We demonstrate that wild type EPEC infection disassembles microtubules and induces the progressive movement of occludin away from the membrane and into the cytosol. Deletion of espG1/G2 attenuates both of these phenotypes. In addition, EPEC infection impedes barrier recovery from calcium switch, suggesting that inhibition of TJ restoration, not merely disruption, prolongs barrier loss. TJs recover more rapidly following infection with ΔespG1/G2 than with wild type EPEC, demonstrating that EspG1/G2 perpetuate barrier loss. Although EspG regulates ADP-ribosylation factor (ARF) and p21-activated kinase (PAK), these activities are not necessary for microtubule destruction or perturbation of TJ structure and function. These data strongly support a role for EspG1/G2 and its associated effects on microtubules in delaying the recovery of damaged tight junctions caused by EPEC infection.

Microtubules are required for efficient epithelial tight junction homeostasis and restoration.

Epithelial tight junctions are critical for creating a barrier yet allowing paracellular transport. Although it is well established that the actin cytoskeleton is critical for preserving the dynamic organization of the tight junction and maintaining normal tight junction protein recycling, contributions of microtubules to tight junction organization and function remain undefined. The aim of this study is to determine the role of microtubules in tight junction homeostasis and restoration. Our data demonstrate that occludin traffics on microtubules and that microtubule disruption perturbs tight junction structure and function. Microtubules are also shown to be required for restoring barrier function following Ca(2+) chelation and repletion. These processes are mediated by proteins participating in microtubule minus-end-directed trafficking but not plus-end-directed trafficking. These studies show that microtubules participate in the preservation of epithelial tight junction structure and function and play a vital role in tight junction restoration, thus expanding our understanding of the regulation of tight junction physiology.

Adipose inflammation and macrophage infiltration after binge ethanol and burn injury.

BACKGROUND: Ethanol (EtOH) exposure prior to traumatic injury, such as a burn, elevates systemic and local inflammatory responses and increases morbidity and mortality. Adipose is a large tissue mass that is often inflamed during obesity or other stresses, which disturbs metabolic homeostasis. To date, there has been little investigation into the inflammatory response of adipose tissue after combined EtOH exposure and burn injury. METHODS: Two EtOH exposure regimens were utilized to examine the role of inflammation in adipose tissue after EtOH and burn injury. Mice were either given a single or episodic binge exposure to EtOH or saline followed by scald (burn) or sham injury 30 minutes later. Twenty-four hours post injury, serum and adipose tissue were collected for assessment of inflammatory mediators. RESULTS: Single binge EtOH alone induced no inflammation in adipose when compared with sham vehicle-treated mice. However, single binge EtOH followed by burn injury induced significant elevations in mRNA and protein concentrations of pro-inflammatory mediators interleukin-6 (IL-6), KC, and monocyte chemoattractant protein 1 compared with either insult alone or sham vehicle group. Additionally, EtOH exposure and burn injury significantly blunted inducible nitric oxide synthase (iNOS), indicating a complex inflammatory response. Episodic binge EtOH exposure followed by burn injury exacerbated the postburn adipose inflammatory response. The magnitude of the episodic binge-induced inflammatory parameters postburn were 2- to 5-fold greater than the response detected after a single exposure of EtOH, indicating EtOH-induced potentiation of burn-induced inflammatory response. Finally, inflammatory loci and crown-like structures in adipose were significantly increased by episodic binge EtOH and burn injury. CONCLUSIONS: This is the first report of binge and burn-induced crown-like structure formation. Evidence presented herein suggests an important role for alcohol and burn as an additional mediator of adipose inflammation in postburn injury, a common complication in burn patients.

Epigenetic targets for reversing immune defects caused by alcohol exposure.

Alcohol consumption alters factors that modify gene expression without changing the DNA code (i.e., epigenetic modulators) in many organ systems, including the immune system. Alcohol enhances the risk for developing several serious medical conditions related to immune system dysfunction, including acute respiratory distress syndrome (ARDS), liver cancer, and alcoholic liver disease (ALD). Binge and chronic drinking also render patients more susceptible to many infectious pathogens and advance the progression of HIV infection by weakening both innate and adaptive immunity. Epigenetic mechanisms play a pivotal role in these processes. For example, alcohol-induced epigenetic variations alter the developmental pathways of several types of immune cells (e.g., granulocytes, macrophages, and T-lymphocytes) and through these and other mechanisms promote exaggerated inflammatory responses. In addition, epigenetic mechanisms may underlie alcohol's ability to interfere with the barrier functions of the gut and respiratory systems, which also contribute to the heightened risk of infections. Better understanding of alcohol's effects on these epigenetic processes may help researchers identify new targets for the development of novel medications to prevent or ameliorate alcohol's detrimental effects on the immune system.

Intestinal barrier disruption as a cause of mortality in combined radiation and burn injury.

Nuclear disaster associated with combined radiation injury (CRI) and trauma or burns results in higher mortality than component injuries. Early death is caused by sequelae of gastrointestinal (GI) leakiness such as bacterial translocation and shock. We developed a murine model to characterize GI injury after CRI and determine the extent of barrier disruption. Animals received radiation (5.5 Gy) alone or with 15% total body surface area (TBSA) scald burn and were euthanized at 24, 48, and 72 h. Mesenteric lymph node homogenate was plated on tryptic soy agar to assess for bacterial translocation. Tight junction protein, occludin, was characterized by Western blot and immunofluorescence. Intestinal histology was evaluated, and apoptosis was quantified using histone-associated DNA fragmentation enzyme-linked immunosorbent assay and Western blot for caspase-3 and caspase-8. At 72 h, a 100-fold increase in bacterial growth after CRI was observed. Occludin colocalization was reduced by radiation exposure, with largest differences in CRI at 24 and 48 h. Histopathology exhibited increased apoptosis in radiation alone and CRI animals at 24 and 48 h (P < 0.05). Further evidence of apoptotic activity in CRI was seen at 48 h, with 3-fold increases in enzyme-linked immunosorbent assay detection relative to all groups and caspase-8 activity relative to radiation alone and sham (P < 0.05). Prolonged epithelial apoptosis and disruption of tight junctions likely contribute to gut leakiness after CRI. Subsequent bacterial translocation to mesenteric lymph node potentially leads to sepsis and death and could serve as a target for mitigating agents to improve survival from CRI.

Pulmonary inflammation after ethanol exposure and burn injury is attenuated in the absence of IL-6.

Alcohol consumption leads to an exaggerated inflammatory response after burn injury. Elevated levels of interleukin-6 (IL-6) in patients are associated with increased morbidity and mortality after injury, and high systemic and pulmonary levels of IL-6 have been observed after the combined insult of ethanol exposure and burn injury. To further investigate the role of IL-6 in the pulmonary inflammatory response, we examined leukocyte infiltration and cytokine and chemokine production in the lungs of wild-type and IL-6 knockout mice given vehicle or ethanol (1.11 g/kg) and subjected to a sham or 15% total body surface area burn injury. Levels of neutrophil infiltration and neutrophil chemoattractants were increased to a similar extent in wild-type and IL-6 knockout mice 24 h after burn injury. When ethanol exposure preceded the burn injury, however, a further increase of these inflammatory markers was seen only in the wild-type mice. Additionally, signal transducer and activator of transcription-3 (STAT3) phosphorylation did not increase in response to ethanol exposure in the IL-6 knockout mice, in contrast to their wild-type counterparts. Visual and imaging analysis of alveolar wall thickness supported these findings and similar results were obtained by blocking IL-6 with antibody. Taken together, our data suggest a causal relationship between IL-6 and the excessive pulmonary inflammation observed after the combined insult of ethanol and burn injury.

Anti-IL-6 antibody treatment but not IL-6 knockout improves intestinal barrier function and reduces inflammation after binge ethanol exposure and burn injury.

Interleukin 6 (IL-6) is an inflammatory cytokine known to be elevated in chronic diseases and after insults such as trauma and infection. Although necessary for the development of B cells and Th17 cells, IL-6, at elevated levels, can also cause tissue damage and lead to a rise in inflammation. Previous work in our laboratory has shown that IL-6 is increased both systemically and in multiple organ systems including the ileum after ethanol exposure and burn injury. As this combined insult causes elevated intestinal morphological damage, tight junction protein localization alterations, and phosphorylated myosin light chain levels, we sought to determine the role of IL-6 in these intestinal responses using a model of binge ethanol exposure and burn injury. Interleukin 6 antibody treatment after the combined insult reduced morphological changes in the ileum, bacterial translocation, and phosphorylated myosin light chain levels relative to either injury alone. Zonula occludens protein 1 and occludin localization was also reestablished in wild-type mice given IL-6 antibody after ethanol and burn. Interleukin 6-knockout mice given ethanol and burn injury also had reduced intestinal damage; however, no changes in bacterial translocation or tight junction protein localization were observed as compared with similarly treated wild-type mice. These data suggest that IL-6 may have a role in intestinal tissue damage observed after the combined insult of binge ethanol exposure and burn injury, although complete loss of IL-6 does not seem to be beneficial in this model. Modulation of IL-6 may present a new option for preventing intestinal damage and associated inflammation after a combined insult of ethanol exposure and burn injury.

Inhibition of long myosin light-chain kinase activation alleviates intestinal damage after binge ethanol exposure and burn injury.

Laboratory evidence suggests that intestinal permeability is elevated following either binge ethanol exposure or burn injury alone, and this barrier dysfunction is further perturbed when these insults are combined. We and others have previously reported a rise in both systemic and local proinflammatory cytokine production in mice after the combined insult. Knowing that long myosin light-chain kinase (MLCK) is important for epithelial barrier maintenance and can be activated by proinflammatory cytokines, we examined whether inhibition of MLCK alleviated detrimental intestinal responses seen after ethanol exposure and burn injury. To accomplish this, mice were given vehicle or a single binge ethanol exposure followed by a sham or dorsal scald burn injury. Following injury, one group of mice received membrane permeant inhibitor of MLCK (PIK). At 6 and 24 h postinjury, bacterial translocation and intestinal levels of proinflammatory cytokines were measured, and changes in tight junction protein localization and total intestinal morphology were analyzed. Elevated morphological damage, ileal IL-1ß and IL-6 levels, and bacterial translocation were seen in mice exposed to ethanol and burn injury relative to either insult alone. This increase was not seen in mice receiving PIK after injury. Ethanol-exposed and burn-injured mice had reduced zonula occludens protein-1 and occludin localization to the tight junction relative to sham-injured mice. However, the observed changes in junctional complexes were not seen in our PIK-treated mice following the combined insult. These data suggest that MLCK activity may promote morphological and inflammatory responses in the ileum following ethanol exposure and burn injury.

Alcohol and epigenetic changes: summary of the 2011 Alcohol and Immunology Research Interest Group (AIRIG) meeting.

On November 18, 2011, the 16th annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held at Loyola University Medical Center in Maywood, Illinois. The focus of this year's meeting was alcohol's effect on epigenetic changes and possible outcomes induced by these changes. Two sessions, which consisted of talks from invited speakers as well as presentations of selected abstracts, were held in addition to a poster session. Participants presented information on alcohol-induced alterations in histone modifications and gene expression along with immunologic responses to alcohol. Speakers shared new research specifically on histone deacetylase enzyme expression and modifications due to alcohol and the downstream effect of these modifications may have on gene expression and tissue damage. Additional studies suggested that alcohol exacerbates inflammation when combined with other insults such as infection, trauma, inhalation injury, and disease.

Alcohol and inflammation and infection: clinical and experimental systems--summary of the 2010 Alcohol and Immunology Research Interest Group Meeting.

The 15th annual meeting of the Alcohol and Immunology Research Interest Group was held on November 19, 2010, at Loyola University Medical Center in Maywood, IL. This year, the focus of the meeting was on alcohol's effect on the immune system in both clinical and experimental systems. The event consisted of three sessions, which featured plenary talks from invited speakers along with oral presentations from selected abstracts, in addition to a poster session. Participants presented a variety of information on ethanol-induced effects on infection susceptibility and resolution, oxidative stress, and organ inflammation. Specifically, speakers presented new insights on the mechanism of alcohol-mediated deleterious effects in the lung, liver, skin, and neuroendocrine system, as well as on immune cells in both in vivo and in vitro systems. Additional oral presentations suggested possible mechanisms of how alcohol-induced reactive oxygen species promote immune dysregulation both locally and systemically.

Decreased pulmonary inflammation following ethanol and burn injury in mice deficient in TLR4 but not TLR2 signaling.

BACKGROUND: Clinical and laboratory evidence suggests that alcohol consumption prior to burn injury leads to dysregulated immune function and subsequent higher rates of morbidity and mortality. Our laboratory previously observed higher levels of pro-inflammatory cytokines and leukocyte infiltration in the lungs of mice following ethanol and burn injury. To understand the mechanism of the increased inflammatory response, we looked at different signaling initiators of inflammation including toll-like receptors 2 and 4 (TLR2 and 4) pathways. METHODS: Wild-type, TLR2, and TLR4 knockout mice were treated with vehicle or a single binge dose of ethanol (1.11 g/kg) and subsequently given a sham or burn injury. Twenty-four hours postinjury, systemic and pulmonary levels of pro-inflammatory cytokines were quantified, and differences in neutrophil infiltration were determined by histological examination. RESULTS: Higher numbers of neutrophils were observed in the lungs of wild-type mice following the combined insult of ethanol and burn injury relative to either injury alone. This increase in leukocyte accumulation was absent in the TLR4 knockout mice. Circulating levels of IL-6 and tumor necrosis factor-α were also elevated in wild-type mice but not in TLR4 knockout mice. Consistent with these findings, pulmonary levels of KC and IL-6 were increased in wild-type mice following burn and ethanol compared to burn injury alone as well as to their TLR4 knockout counterparts. In contrast, TLR2 knockout mice displayed similar levels, to wild-type mice, of neutrophil infiltration as well as IL-6 and KC in the lung. CONCLUSIONS: These data suggest that TLR4 signaling is a crucial contributory component in the exuberant inflammation after ethanol and burn injury. However, TLR2 does not appear to play a vital role in the aberrant pulmonary inflammation.

Touchtone promotes survival of embryonic melanophores in zebrafish.

An outstanding problem in the study of vertebrate development is the identification of the genes that direct neural crest precursor cells to adopt and maintain specific differentiated cell fates. In an effort to identify such genes, we have carried out a mutagenesis screen in zebrafish and isolated mutants that lack neural crest-derived melanophores. In this manuscript we describe the phenotype of one such mutant, touchtone(b722) (tct), and the map position of the gene it defines. Analysis of expression of dopachrome tautomerase (dct) and microphthalmia (mitfa) suggests that melanophore precursors are specified normally in homozygous tct mutants. However, differentiated melanophores are pale, small, and about half of them have disappeared by 48 h of development, apparently by cell death. We show that melanophores require Tct function cell autonomously. Signals from the receptor tyrosine kinase receptor C-kit are essential for survival of melanophores in zebrafish and mammals. However, differences in the phenotypes of tct and c-kit homozygous mutants, and an absence of interaction between c-kit and tct heterozygotes, suggest that Tct functions independently of the C-kit pathway. Other neural crest derivatives, including other pigment cell types, appear normal in tct mutants. Interestingly, tct mutant embryos undergo a temporary period of near complete paralyzis during the second day of development, although markers of axons of motor and sensory neurons look normal in this period. A fraction of tct(b722) mutants survive to adulthood, but mutant adults are small, indicating a role for Tct in post-larval growth. The tct gene maps to a small interval near a telomere of chromosome 18. Thus, we have identified a zebrafish gene that when mutated produces semi-viable offspring and that may serve as a model of human diseases that have both pigmentation and neurological symptoms.