Recent alcohol intake impacts microbiota in adult burn patients.

Alcohol use is associated with an increased incidence of negative health outcomes in burn patients due to biological mechanisms that include a dysregulated inflammatory response and increased intestinal permeability. This study used phosphatidylethanol (PEth) in blood, a direct biomarker of recent alcohol use, to investigate associations between a recent history of alcohol use and the fecal microbiota, short chain fatty acids, and inflammatory markers in the first week after a burn injury for nineteen participants. Burn patients were grouped according to PEth levels of low or high and differences in the overall fecal microbial community were observed between these cohorts. Two genera that contributed to the differences and had higher relative abundance in the low PEth burn patient group were Akkermansia, a mucin degrading bacteria that improves intestinal barrier function, and Bacteroides, a potentially anti-inflammatory bacteria. There was no statistically significant difference between levels of short chain fatty acids or intestinal permeability across the two groups. To our knowledge, this study represents the first report to evaluate the effects of burn injury and recent alcohol use on early post burn microbiota dysbiosis, inflammatory response, and levels of short chain fatty acids. Future studies in this field are warranted to better understand the factors associated with negative health outcomes and develop interventional trials.

Emerging concepts in alcohol, infection & immunity: A summary of the 2023 alcohol and immunology research interest group (AIRIG) meeting.

On December 8th 2023, the annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held at the University of Colorado Anschutz Medical Campus in Aurora, Colorado. The 2023 meeting focused broadly on how acute and chronic alcohol exposure leads to immune dysregulation, and how this contributes to damage in multiple tissues and organs. These include impaired lung immunity, intestinal dysfunction, autoimmunity, the gut-Central Nervous System (CNS) axis, and end-organ damage. In addition, diverse areas of alcohol research covered multiple pathways behind alcohol-induced cellular dysfunction, including inflammasome activation, changes in miRNA expression, mitochondrial metabolism, gene regulation, and transcriptomics. Finally, the work presented at this meeting highlighted novel biomarkers and therapeutic interventions for patients suffering from alcohol-induced organ damage.

Multi-omics analysis of alcohol effects on the liver in young and aged mice.

Excessive alcohol consumption has detrimental effects on the entire organism, especially on the liver. The toxicity is partly dependent on age, as older individuals metabolize alcohol more slowly leading to increased cellular injury. This study aimed to investigate the effects of moderate binge drinking on the liver of young and aged mice in a genome-wide multi-omics approach. We determined DNA methylation (DNAm) using the Illumina MouseMethylation array and gene expression by RNA sequencing in 18 female Balb/c mice in a 2 × 2 design. The animals underwent three moderate binge drinking cycles (ethanol vs. vehicle) and liver tissue was harvested at 4 or 19 months of age. We tested differential gene expression (DE) and DNAm associated with ethanol intake in linear models separately in young and aged mice, performed enrichment analyses for pathways and GWAS signatures of problematic alcohol use, and analysed the overlap of DNAm and gene expression. We observed DE in young and aged animals and substantial overlap in genes such as Bhlhe40, Klf10, and Frmd8. DE genes in aged animals were enriched for biological processes related to alcohol metabolism, inflammation, liver fibrosis, and GWAS signatures of problematic alcohol use. We identified overlapping signatures from DNAm and gene expression, for example, Frmd8 in aged and St6galnac4 in young mice. This study offers converging evidence of novel age-related targets in a moderate alcohol consumption model highlighting dysregulations in genes related to alcohol metabolism, inflammation, and liver fibrosis. Future studies are needed to confirm these results and elucidate the underlying mechanisms.

Ethanol exacerbates pulmonary complications after burn injury in mice, regardless of frequency of ethanol exposures.

Burn injuries are associated with significant morbidity and mortality, and lungs are the most common organ to fail. Interestingly, patients with alcohol intoxication at the time of burn have worse clinical outcomes, including pulmonary complications. Using a clinically relevant murine model, we have previously reported that episodic ethanol exposure before burn exacerbated lung inflammation. Specifically, intoxicated burned mice had worsened pulmonary responses, including increased leukocyte infiltration and heightened levels of CXCL1 and IL-6. Herein, we examined whether a single binge ethanol exposure before scald burn injury yields similar pulmonary responses. C57BL/6 male mice were given ethanol (1.2 g/kg) 30 min before a 15 % total body surface area burn. These mice were compared to a second cohort given episodic ethanol binge for a total of 6 days (3 days ethanol, 4 days rest, 3 days ethanol) prior to burn injury. 24 h after burn, histopathological examination of lungs were performed. In addition, survival, and levels of infiltrating leukocytes, CXCL1, and IL-6 were quantified. Episodic and single ethanol exposure before burn decreased survival compared to burn only mice and sham vehicle mice, respectively (p < 0.05). However, no difference in survival was observed between burned mice with single and episodic ethanol binge. Examination of H&E-stained lung sections revealed that regardless of ethanol binge frequency, intoxication prior to burn worsened pulmonary inflammation, evidenced by elevated granulocyte accumulation and congestion, relative to burned mice without any ethanol exposure. Levels of infiltrating granulocyte in the lungs were significantly higher in burned mice with both episodic and single ethanol intoxication, compared to burn injury only (p < 0.05). In addition, there was no difference in the granulocyte count between single and ethanol binge mice with burn injury. Neutrophil chemoattractant CXCL1 levels in the lung were similarly increased following single and episodic ethanol exposure prior to burn compared to burn alone (22-fold and 26-fold respectively, p < 0.05). Lastly, we assessed pulmonary IL-6, which revealed that irrespective of frequency, ethanol exposure combined with burn injury raised pro-inflammatory cytokine IL-6 in the lungs relative to burn mice. Again, we did not find any difference in the amount of IL-6 in lungs of burned mice with single and episodic ethanol intoxication. Taken altogether, these data demonstrate that both single and episodic exposure to ethanol prior to burn injury similarly worsens pulmonary inflammation. These results suggest that ethanol-induced exacerbation of the pulmonary responses to burn injury is due to presence of ethanol at the time of injury rather than longer-term effects of ethanol exposure.

REMOTE BURN INJURY IN AGED MICE INDUCES COLONIC LYMPHOID AGGREGATE EXPANSION AND DYSBIOSIS OF THE FECAL MICROBIOME WHICH CORRELATES WITH NEUROINFLAMMATION.

ABSTRACT: The Earth's population is aging, and by 2050, one of six people will be 65 years or older. Therefore, proper treatment of injuries that disproportionately impact people of advanced age will be more important. Clinical studies reveal people 65 years or older account for 16.5% of all burn injuries and experience higher morbidity, including neurocognitive decline, and mortality that we and others believe are mediated, in part, by heightened intestinal permeability. Herein, we used our clinically relevant model of scald burn injury in young and aged mice to determine whether age and burn injury cooperate to induce heightened colonic damage, alterations to the fecal microbiome, and whether resultant changes in the microbiome correlate with neuroinflammation. We found that aged, burn-injured mice have an increase in colonic lymphoid aggregates, inflammation, and proinflammatory chemokine expression when compared with young groups and sham-injured aged mice. We then performed fecal microbiota sequencing and found a striking reduction in gut protective bacterial taxa, including Akkermansia , in the aged burn group compared with all other groups. This reduction correlated with an increase in serum fluorescein isothiocyanate-Dextran administered by gavage, indicating heightened intestinal permeability. Furthermore, loss of Akkermansia was highly correlated with increased messenger RNA expression of neuroinflammatory markers in the brain, including chemokine ligand 2, TNF-α, CXC motif ligand 1, and S100 calcium-binding protein A8. Finally, we discovered that postburn alterations in the microbiome correlated with measures of strength in all treatment groups, and those that performed better on the rotarod and hanging wire tests had higher abundance of Akkermansia than those that performed worse. Taken together, these findings indicate that loss of protective bacteria after burn injury in aged mice contributes to alterations in the colon, gut leakiness, neuroinflammation, and strength. Therefore, supplementation of protective bacteria, such as Akkermansia , after burn injury in aged patients may have therapeutic benefit.

Alcohol and Immunology: Mechanisms of multi-organ damage. Summary of the 2022 alcohol and Immunology research interest group (AIRIG) meeting.

On October 26th, 2022 the annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held as a satellite symposium at the annual meeting of the Society for Leukocyte Biology in Hawaii. The 2022 meeting focused broadly on the immunological consequences of acute, chronic, and prenatal alcohol exposure and how these contribute to damage in multiple organs and tissues. These included alcohol-induced neuroinflammation, impaired lung immunity, intestinal dysfunction, and decreased anti-microbial and anti-viral responses. In addition, research presented covered multiple pathways behind alcohol-induced cellular dysfunction, including mitochondrial metabolism, cellular bioenergetics, gene regulation, and epigenetics. Finally, the work presented highlighted potential biomarkers and novel avenues of treatment for alcohol-induced organ damage.

Aberrant inflammatory responses in intoxicated burn-injured patients parallel impaired cognitive function.

Burn-injured patients with alcohol use disorder (AUD) have increased morbidity and mortality compared to alcohol-abstaining individuals with similar injuries. It is hypothesized that this is due, in part, to alcohol-induced dysregulation of the systemic inflammatory response, leading to worsened clinical outcomes, including increased susceptibility to infection, and heightened cognitive impairment. To examine the effects of alcohol on inflammatory markers after burn injury, we used multiplex assays to measure a panel of 48 cytokines, chemokines, and growth factors in the plasma of burn patents within 24 h of admission to the University of Colorado Burn Center. Thirty patients were enrolled between July 2018 to February 2020 and were stratified based on presence of AUD and total body surface area (TBSA) burn of ≥20% into four groups: [AUD-, TBSA <20%, N = 12], [AUD+, TBSA <20%, N = 3], [AUD-, TBSA ≥20%, N = 8], [AUD+, TBSA ≥20%, N = 7]. In addition, Confusion Assessment Method (CAM) scores were collected to evaluate patient delirium during the course of hospitalization. Multivariate statistical analysis demonstrated a number of cytokines and other factors that were significantly different between the groups. For example, the anti-inflammatory cytokine interleukin 1 receptor antagonist (IL-1ra) was dampened in the AUD+, TBSA ≥20% cohort with a 75.2% decrease compared to AUD-, TBSA ≥20%, and an 83.9% decrease compared to AUD-, TBSA <20% (p = 0.008). Additionally, plasma levels of the pro-inflammatory mediator CXCL12 (C-X-C motif chemokine ligand 12, also known as stromal cell-derived factor 1, SDF-1) was higher in the AUD + groups (p = 0.03) and similarly, IL-18 levels were greater in AUD+, TBSA ≥20% (p = 0.009). Eotaxin (also known as cytokine CC motif ligand 11, CCL11) was markedly elevated in the AUD+, TBSA ≥20% cohort with a 2.4-fold increase over the AUD-, TBSA ≥20%, and a 1.7-fold rise compared to the AUD-, TBSA <20% cohorts (p = 0.04). Interestingly, there was also a marked rise in CAM + delirium scores (85.7%) among the AUD + patients with TBSA ≥20% (p = 0.02). Not surprisingly, we found that hospital stays increased with AUD+ and larger burns (p = 0.0009). Our findings reveal that burn patients who misuse alcohol have aberrant inflammatory responses that may lead to greater immune dysregulation and worse clinical outcomes.

A critical review of recent knowledge of alcohol's effects on the immunological response in different tissues.

Alcohol misuse contributes to the dysregulation of immune responses and multiorgan dysfunction across various tissues, which are associated with higher risk of morbidity and mortality in people with alcohol use disorders. Organ-specific immune cells, including microglia in the brain, alveolar macrophages in the lungs, and Kupffer cells in the liver, play vital functions in host immune defense through tissue repair and maintenance of homeostasis. However, binge drinking and chronic alcohol misuse impair these immune cells' abilities to regulate inflammatory signaling and metabolism, thus contributing to multiorgan dysfunction. Further complicating these delicate systems, immune cell dysfunction associated with alcohol misuse is exacerbated by aging and gut barrier leakage. This critical review describes recent advances in elucidating the potential mechanisms by which alcohol misuse leads to derangements in host immunity and highlights current gaps in knowledge that may be the focus of future investigations.

Changes in gut microbiome correlate with intestinal barrier dysfunction and inflammation following a 3-day ethanol exposure in aged mice.

Alcohol use among older adults is on the rise. This increase is clinically relevant as older adults are at risk for increased morbidity and mortality from many alcohol-related chronic diseases compared to younger patients. However, little is known regarding the synergistic effects of alcohol and age. There are intriguing data suggesting that aging may lead to impaired intestinal barrier integrity and dysbiosis of the intestinal microbiome, which could increase susceptibility to alcohol's negative effects. To study the effects of alcohol in age we exposed aged and young mice to 3 days of moderate ethanol and evaluated changes in gut parameters. We found that these levels of drinking do not have obvious effects in young mice but cause significant alcohol-induced gut barrier dysfunction and expression of the pro-inflammatory cytokine TNFα in aged mice. Ethanol-induced downregulation of expression of the gut-protective antimicrobial peptides Defa-rs1, Reg3b, and Reg3g was observed in aged, but not young mice. Analysis of the fecal microbiome revealed age-associated shifts in microbial taxa, which correlated with intestinal and hepatic inflammatory gene expression. Taken together, these data demonstrate that age drives microbiome dysbiosis, while ethanol exposure in aged mice induces changes in the expression of antimicrobial genes important for separating these potentially damaging microbes from the intestinal lumen. These changes highlight potential mechanistic targets for prevention of the age-related exacerbation of effects of ethanol on the gut.

Cutaneous burn injury induces neuroinflammation and reactive astrocyte activation in the hippocampus of aged mice.

BACKGROUND: By 2050, one in six people globally will be 65 or older. Confusion and delirium are significant complications after burn injury, especially in the elderly population. The etiology is still unknown, however complications may be driven by pro-inflammatory activation of astrocytes within the hippocampus (HPC) after burn injury. Reduced levels of phosphorylated cyclic-AMP response binding element (pCREB), caused by elevated systemic pro-inflammatory cytokines, could lead to cognitive decline and memory impairment. METHODS: To examine the effects of remote injury on neuroinflammation in advanced age, young and aged mice were subjected to a 15 % total body surface area scald burn or sham injury, and euthanized after 24 h. Expression of ccl2 and tnfa were measured by qPCR in the whole brain and HPC. Astrocyte activation was measured by immunofluorescence within the HPC. pCREB was measured by immunohistochemistry in the dentate gyrus. RESULTS: We saw an 80-fold increase in ccl2 and a 30-fold elevation in tnfa after injury in the whole brain of aged mice compared to young groups and aged sham mice (p < 0.05 and p < 0.05, respectively). Additionally, there was a 30-fold increase in ccl2 within isolated HPC of aged injured mice when compared to sham injured animals (p < 0.05). When investigating specific HPC regions, immunofluorescence staining showed a >20 % rise in glial fibrillary acidic protein (GFAP) positive astrocytes within the cornu ammonis 3 (CA3) of aged injured mice when compared to all other groups (p < 0.05). Lastly, we observed a >20 % decrease in pCREB staining by immunohistochemistry in the dentate gyrus of aged mice compared to young regardless of injury (p < 0.05). CONCLUSIONS: These novel data suggest that remote injury in aged, but not young, mice is associated with neuroinflammation and astrocyte activation within the HPC. These factors, paired with an age related reduction in pCREB, could help explain the increased cognitive decline seen in burn patients of advanced age. To our knowledge, we are the first group to examine the impact of advanced age combined with burn injury on inflammation and astrocyte activation within the brain.

Advanced age is associated with changes in alveolar macrophages and their responses to the stress of traumatic injury.

Alveolar macrophages (AMs) are tissue-resident cells of the lower airways that perform many homeostatic functions critical for pulmonary health and protection against pathogens. However, little is known about the factors that shape AMs during healthy aging. In these studies, we sought to characterize age-related changes in AM phenotype, function, and responses to a physiologic stressor, that is, distal injury. Age was associated with a wide range of changes in cell surface receptor and gene expression by AMs, reflecting a unique alternatively activated phenotype. AMs from aged mice also exhibited markers of cellular senescence along with down-regulation of genes involved in growth and cell cycle pathways relative to young controls. Furthermore, AMs from aged mice showed a stunted transcriptional response to distal injury compared with AMs from young mice. Many changes were found to involve glucocorticoid-regulated genes, and corticosteroid treatment of primary AMs ex vivo revealed diminished transcriptional responses in cells from aged animals. These results demonstrate that there is a complex age-dependent AM phenotype associated with dysregulated stress hormone signaling that may interfere with AM responses to physiologic stressors and could contribute to AM dysfunction and the decline of pulmonary immunity during healthy aging.

Aging, Cutaneous Burn Injury and Multi-Organ Complications: The Role of the Gut.

Advanced age escalates post-burn complications and older burn patients, and even those with relatively minor burns, have worse clinical outcomes after injury. While the mechanism(s) responsible for the compounding effects of age and burn injury have not been defined, in this viewpoint, we highlight the emerging data suggesting that age-mediated impairment of gut barrier integrity and dysbiosis of the fecal microbiome in older subjects may play a role in the heightened multi-organ responses seen in older patients. Studies aimed at exploring the contribution of intestinal dysfunction in age-related exacerbations of post-burn inflammatory responses could highlight novel therapeutic interventions that can be used to treat victims of burns and other traumatic injuries.

New insights into the mechanism of alcohol-mediated organ damage via its impact on immunity, metabolism, and repair pathways: A summary of the 2021 Alcohol and Immunology Research Interest Group (AIRIG) meeting.

On November 19th, 2021, the annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held at Loyola University Chicago Health Sciences Campus in Maywood, Illinois. The 2021 meeting focused on how alcohol misuse is linked to immune system derangements, leading to tissue and organ damage, and how this research can be translated into improving treatment of alcohol-related disease. This meeting was divided into three plenary sessions: the first session focused on how alcohol misuse affects different parts of the immune system, the second session presented research on mechanisms of organ damage from alcohol misuse, and the final session highlighted research on potential therapeutic targets for treating alcohol-mediated tissue damage. Diverse areas of alcohol research were covered during the meeting, from alcohol's effect on pulmonary systems and neuroinflammation to epigenetic changes, senescence markers, and microvesicle particles. These presentations yielded a thoughtful discussion on how the findings can lead to therapeutic treatments for people suffering from alcohol-related diseases.

Ethanol Intoxication Impairs Respiratory Function and Bacterial Clearance and Is Associated With Neutrophil Accumulation in the Lung After Streptococcus pneumoniae Infection.

Alcohol consumption is commonplace in the United States and its prevalence has increased in recent years. Excessive alcohol use is linked to an increased risk of infections including pneumococcal pneumonia, mostly commonly caused by Streptococcus pneumoniae. In addition, pneumonia patients with prior alcohol use often require more intensive treatment and longer hospital stays due to complications of infection. The initial respiratory tract immune response to S. pneumoniae includes the production of pro-inflammatory cytokines and chemokines by resident cells in the upper and lower airways which activate and recruit leukocytes to the site of infection. However, this inflammation must be tightly regulated to avoid accumulation of toxic by-products and subsequent tissue damage. A majority of previous work on alcohol and pneumonia involve animal models utilizing high concentrations of ethanol or chronic exposure and offer conflicting results about how ethanol alters immunity to pathogens. Further, animal models often employ a high bacterial inoculum which may overwhelm the immune system and obscure results, limiting their applicability to the course of human infection. Here, we sought to determine how a more moderate ethanol exposure paradigm affects respiratory function and innate immunity in mice after intranasal infection with 104 colony forming units of S. pneumoniae. Ethanol-exposed mice displayed respiratory dysfunction and impaired bacterial clearance after infection compared to their vehicle-exposed counterparts. This altered response was associated with increased gene expression of neutrophil chemokines Cxcl1 and Cxcl2 in whole lung homogenates, elevated concentrations of circulating granulocyte-colony stimulating factor (G-CSF), and higher neutrophil numbers in the lung 24 hours after infection. Taken together, these findings suggest that even a more moderate ethanol consumption pattern can dramatically modulate the innate immune response to S. pneumoniae after only 3 days of ethanol exposure and provide insight into possible mechanisms related to the compromised respiratory immunity seen in alcohol consumers with pneumonia.

Age-Related Intestinal Dysbiosis and Enrichment of Gut-specific Bacteria in the Lung Are Associated With Increased Susceptibility to Streptococcus pneumoniae Infection in Mice.

The portion of the global population that is over the age of 65 is growing rapidly and this presents a number of clinical complications, as the aged population is at higher risk for various diseases, including infection. For example, advanced age is a risk factor for heightened morbidity and mortality following infection with Streptococcus pneumoniae. This increased vulnerability is due, at least in part, to age-related dysregulation of the immune response, a phenomenon termed immunosenescence. However, our understanding of the mechanisms influencing the immunosenescent state and its effects on the innate immune response to pneumonia remain incomplete. Recently, a role for the gut microbiome in age-specific alterations in immunity has been described. Here, we utilized a murine model of intranasal Streptococcus pneumoniae infection to investigate the effects of age on both the innate immune response and the intestinal microbial populations after infection. In aged mice, compared to their younger counterparts, infection with Streptococcus pneumoniae led to increased mortality, impaired lung function and inadequate bacterial control. This poor response to infection was associated with increased influx of neutrophils into the lungs of aged mice 24 h after infection. The exacerbated pulmonary immune response was not associated with increased pro-inflammatory cytokines in the lung compared to young mice but instead heightened expression of immune cell recruiting chemokines by lung neutrophils. Bacterial 16S-rRNA gene sequencing of the fecal microbiome of aged and young-infected mice revealed expansion of Enterobacteriaceae in the feces of aged, but not young mice, after infection. We also saw elevated levels of gut-derived bacteria in the lung of aged-infected mice, including the potentially pathogenic symbiote Escherichia coli. Taken together, these results reveal that, when compared to young mice, Streptococcus pneumoniae infection in age leads to increased lung neutrophilia along with potentially pathogenic alterations in commensal bacteria and highlight potential mechanistic targets contributing to the increased morbidity and mortality observed in infections in age.

Age and Injury Size Influence the Magnitude of Fecal Dysbiosis in Adult Burn Patients.

Clinical studies have demonstrated that age 50 years or older is an independent risk factor associated with poor prognosis after burn injury, the second leading cause of traumatic injuries in the aged population. While mechanisms driving age-dependent postburn mortality are perplexing, changes in the intestinal microbiome, may contribute to the heightened, dysregulated systemic response seen in aging burn patients. The fecal microbiome from 22 patients admitted to a verified burn center from July 2018 to February 2019 was stratified based on the age of 50 years and total burn surface area (TBSA) size of ≥10%. Significant differences (P = .014) in overall microbiota community composition (ie, beta diversity) were measured across the four patient groups: young <10% TBSA, young ≥10% TBSA, older <10% TBSA, and older ≥10% TBSA. Differences in beta diversity were driven by %TBSA (P = .013) and trended with age (P = .087). Alpha diversity components, richness, evenness, and Shannon diversity were measured. We observed significant differences in bacterial species evenness (P = .0023) and Shannon diversity (P = .0033) between the groups. There were significant correlations between individual bacterial species and levels of short-chain fatty acids. Specifically, levels of fecal butyrate correlated with the presence of Enterobacteriaceae, an opportunistic gut pathogen, when elevated in burn patients lead to worsen outcomes. Overall, our findings reveal that age-specific changes in the fecal microbiome following burn injuries may contribute to immune system dysregulation in patients with varying TBSA burns and potentially lead to worsened clinical outcomes with heightened morbidity and mortality.

Advanced age exacerbates intestinal epithelial permeability after burn injury in mice.

BACKGROUND: Advanced age is an independent risk factor for morbidity and mortality after burn injury. Following burn, the intestines can become permeable leading to the leakage of bacteria and their products from the lumen of the ileum to the portal and systemic circulation. Here, we sought to determine the effects of advanced age on intestinal permeability post burn injury and assess intestinal inflammatory biomarkers. METHODS: Young (4-5 months) and aged (18-22 months) female BALB/cBy mice were subjected to a 12-15% total body surface area (TBSA) sham or burn injury. 24 h after injury, mice were euthanized, and organs collected. Colony-forming units (CFU) were counted from plated mesenteric lymph nodes (MLN). Gene expression of ileal tight junctional proteins, occludin and zonula occludens 1 (ZO-1), in addition to ileal damage associated molecular pattern (DAMP) proteins, S100A8 and S100A9, as well as ileal inflammatory markers IL-6 and TNF-α were measured by qPCR. Intestinal cell death was measured by ELISA. Intestinal permeability was determined by FITC fluorescence in serum; 4kD FITC-dextran was given by oral gavage 3 h before euthanasia. RESULTS: Aged mice subjected to burn injury had increased intestinal permeability as evidenced by a 5.8-fold higher level of FITC-dextran in their serum when compared to all other groups (p < 0.05). In addition, aged burn-injured mice exhibited heightened bacterial accumulation in the MLN with a 15.5-fold increase over all other groups (p < 0.05). Histology of ileum failed to show differences in villus length among all groups. Analysis of ileal tight junctional proteins and inflammatory marker gene expression revealed no difference in Ocln, Tjp1, Il6, or Tnf expression among all groups, but 2.3 and 2.9-fold upregulation of S100a8 and S100a9, respectively, in aged burn-injured mice relative to both young groups and aged sham-injured mice (p < 0.05). Lastly, cell death in the ileum was elevated more than two-fold in aged burn-injured mice relative to young animals regardless of injury (p < 0.05). CONCLUSIONS: These data demonstrate that advanced age exacerbates intestinal epithelial permeability after burn injury. Heightened apoptosis may be responsible for the elevated intestinal leakiness and accumulation of bacteria in mesenteric lymph nodes. In addition, S100a8/9 may serve as a biomarker of elevated inflammation within the intestine.

A novel murine model of multi-day moderate ethanol exposure reveals increased intestinal dysfunction and liver inflammation with age.

BACKGROUND: There are currently > 600 million people over the age of 65 globally and this number is expected to double by the year 2050. Alcohol use among this population is on the rise, which is concerning as aging is associated with increased risk for a number of chronic illnesses. As most studies investigating the effects of alcohol have focused on young/middle-aged populations, there is a dearth of information regarding the consequences of alcohol use in older consumers. In addition, most murine ethanol models have concentrated on exposure to very high levels of ethanol, while the vast majority of elderly drinkers do not consume alcohol in excess; instead, they drink on average 2 alcoholic beverages a day, 3-4 days a week. METHODS: We designed a murine model of aging and moderate ethanol consumption to determine if the deleterious effects of alcohol on the gut-liver axis are exacerbated in aged, relative to younger, animals. Aged and young mice were exposed to a multi-day moderate exposure ethanol regimen for 4 weeks and changes in gut permeability along with intestinal tight junction protein and antimicrobial peptide gene expression were measured. In addition, hepatic inflammation was assessed by histological analysis, inflammatory gene expression and flow cytometric analysis of inflammatory infiltrate. RESULTS: Our results reveal that in aged, but not young mice, moderate ethanol exposure yielded significantly worsened intestinal permeability, including increased bacterial translocation from the gut, elevated serum iFABP and leakage of FITC-dextran from the gut. Interestingly, moderate ethanol exposure in young animals led to gut protective transcriptional changes in the ileum while this protective response was blunted in aged mice. Finally, moderate ethanol exposure in aged mice also resulted in marked inflammatory changes in the liver. CONCLUSIONS: These results demonstrate that aged mice are more susceptible to ethanol-induced gut barrier dysfunction and liver inflammation, even at moderate doses of ethanol. This increased vulnerability to ethanol's gastrointestinal effects has important implications for alcohol use in the aging population. Future studies will explore whether improving intestinal barrier function can reverse these age-related changes.

Oxidized Low-Density Lipoprotein Drives Dysfunction of the Liver Lymphatic System.

BACKGROUND AND AIMS: As the incidence of nonalcoholic steatohepatitis (NASH) continues to rise, understanding how normal liver functions are affected during disease is required before developing novel therapeutics which could reduce morbidity and mortality. However, very little is understood about how the transport of proteins and cells from the liver by the lymphatic vasculature is affected by inflammatory mediators or during disease. METHODS: To answer these questions, we utilized a well-validated mouse model of NASH and exposure to highly oxidized low density lipoprotein (oxLDL). In addition to single cell sequencing, multiplexed immunofluorescence and metabolomic analysis of liver lymphatic endothelial cells (LEC)s we evaluated lymphatic permeability and transport both in vitro and in vivo. RESULTS: Confirming similarities between human and mouse liver lymphatic vasculature in NASH, we found that the lymphatic vasculature expands as disease progresses and results in the downregulation of genes important to lymphatic identity and function. We also demonstrate, in mice with NASH, that fluorescein isothiocyanate (FITC) dextran does not accumulate in the liver draining lymph node upon intrahepatic injection, a defect that was rescued with therapeutic administration of the lymphatic growth factor, recombinant vascular endothelial growth factor C (rVEGFC). Similarly, exposure to oxLDL reduced the amount of FITC-dextran in the portal draining lymph node and through an LEC monolayer. We provide evidence that the mechanism by which oxLDL impacts lymphatic permeability is via a reduction in Prox1 expression which decreases lymphatic specific gene expression, impedes LEC metabolism and reorganizes the highly permeable lymphatic cell-cell junctions which are a defining feature of lymphatic capillaries. CONCLUSIONS: We identify oxLDL as a major contributor to decreased lymphatic permeability in the liver, a change which is consistent with decreased protein homeostasis and increased inflammation during chronic liver disease.