Reducing the excessive inflammation after burn injury in aged mice by maintaining a healthier intestinal microbiome.

One in six people are projected to be 65 years or older by 2050. As the population ages, better treatments for injuries that disproportionately impact the aged population will be needed. Clinical studies show that people aged 65 and older experience higher rates of morbidity and mortality after burn injury, including a greater incidence of pulmonary complications when compared to younger burn injured adults, which we and others believe is mediated, in part, by inflammation originating in the intestines. Herein, we use our clinically relevant model of scald burn injury in young and aged mice to determine whether cohousing aged mice with young mice or giving aged mice oral gavage of fecal material from young mice is sufficient to alter the microbiome of the aged mice and protect them from inflammation in the ileum and the lungs. Aged burn injured mice have less DNA expression of Bacteroidetes in the feces and an unhealthy Firmicutes/Bacteroidetes ratio. Both Bacteroidetes and the ratio of these two phyla are restored in aged burn injured by prior cohousing for a month with younger mice but not fecal transfer from young mice. This shift in the microbiome coincides with heightened expression of danger-associated molecular patterns (DAMP), and pro-inflammatory cytokine interleukin-6 (il6) in the ileum and lung of aged, burn injured mice, and heightened antimicrobial peptide camp in the lung. Cohousing reverses DAMP expression in the ileum and lung, and cathelicidin-related antimicrobial peptide protein (camp) in the lung, while fecal transfer heightened DAMPs while reducing camp in the lung, and also increased IL-6 protein in the lungs. These results highlight the importance of the intestinal microbiome in mediating inflammation within the gut-lung axis, giving insights into potential future treatments in the clinic.

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.

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.

Dietary Lipids Differentially Shape Nonalcoholic Steatohepatitis Progression and the Transcriptome of Kupffer Cells and Infiltrating Macrophages.

A crucial component of nonalcoholic fatty liver disease (NAFLD) pathogenesis is lipid stress, which may contribute to hepatic inflammation and activation of innate immunity in the liver. However, little is known regarding how dietary lipids, including fat and cholesterol, may facilitate innate immune activation in vivo. We hypothesized that dietary fat and cholesterol drive NAFLD progression to steatohepatitis and hepatic fibrosis by altering the transcription and phenotype of hepatic macrophages. This hypothesis was tested by using RNA-sequencing methods to characterize and analyze sort-purified hepatic macrophage populations that were isolated from mice fed diets with varying amounts of fat and cholesterol. The addition of cholesterol to a high-fat diet triggered hepatic pathology reminiscent of advanced nonalcoholic steatohepatitis (NASH) in humans characterized by signs of cholesterol dysregulation, generation of oxidized low-density lipoprotein, increased recruitment of hepatic macrophages, and significant fibrosis. RNA-sequencing analyses of hepatic macrophages in this model revealed that dietary cholesterol induced a tissue repair and regeneration phenotype in Kupffer cells (KCs) and recruited infiltrating macrophages to a greater degree than fat. Furthermore, comparison of diseased KCs and infiltrating macrophages revealed that these two macrophage subsets are transcriptionally diverse. Finally, direct stimulation of murine and human macrophages with oxidized low-density lipoprotein recapitulated some of the transcriptional changes observed in the RNA-sequencing study. These findings indicate that fat and cholesterol synergize to alter macrophage phenotype, and they also challenge the dogma that KCs are purely proinflammatory in NASH. Conclusion: This comprehensive view of macrophage populations in NASH indicates mechanisms by which cholesterol contributes to NASH progression and identifies potential therapeutic targets for this common disease.

How an alloreactive T-cell receptor achieves peptide and MHC specificity.

T-cell receptor (TCR) allorecognition is often presumed to be relatively nonspecific, attributable to either a TCR focus on exposed major histocompatibility complex (MHC) polymorphisms or the degenerate recognition of allopeptides. However, paradoxically, alloreactivity can proceed with high peptide and MHC specificity. Although the underlying mechanisms remain unclear, the existence of highly specific alloreactive TCRs has led to their use as immunotherapeutics that can circumvent central tolerance and limit graft-versus-host disease. Here, we show how an alloreactive TCR achieves peptide and MHC specificity. The HCV1406 TCR was cloned from T cells that expanded when a hepatitis C virus (HCV)-infected HLA-A2- individual received an HLA-A2+ liver allograft. HCV1406 was subsequently shown to recognize the HCV nonstructural protein 3 (NS3):1406-1415 epitope with high specificity when presented by HLA-A2. We show that NS3/HLA-A2 recognition by the HCV1406 TCR is critically dependent on features unique to both the allo-MHC and the NS3 epitope. We also find cooperativity between structural mimicry and a crucial peptide "hot spot" and demonstrate its role, along with the MHC, in directing the specificity of allorecognition. Our results help explain the paradox of specificity in alloreactive TCRs and have implications for their use in immunotherapy and related efforts to manipulate TCR recognition, as well as alloreactivity in general.

Perilipin-2 promotes obesity and progressive fatty liver disease in mice through mechanistically distinct hepatocyte and extra-hepatocyte actions.

KEY POINTS: Wild-type mice and mice with hepatocyte-specific or whole-body deletions of perilipin-2 (Plin2) were used to define hepatocyte and extra-hepatocyte effects of altered cellular lipid storage on obesity and non-alcoholic fatty liver disease (NAFLD) pathophysiology in a Western-diet (WD) model of these disorders. Extra-hepatocyte actions of Plin2 are responsible for obesity, adipose inflammation and glucose clearance abnormalities in WD-fed mice. Hepatocyte and extra-hepatic actions of Plin2 mediate fatty liver formation in WD-fed mice through distinct mechanisms. Hepatocyte-specific actions of Plin2 are primary mediators of immune cell infiltration and fibrotic injury in livers of obese mice. ABSTRACT: Non-alcoholic fatty liver disease (NAFLD) is an obesity- and insulin resistance-related metabolic disorder with progressive pathology. Perilipin-2 (Plin2), a ubiquitously expressed cytoplasmic lipid droplet scaffolding protein, is hypothesized to contribute to NAFLD in humans and rodent models through effects on cellular lipid metabolism. In this study, we delineate hepatocyte-specific and extra-hepatocyte Plin2 mechanisms regulating the effects of obesity and insulin resistance on NAFLD pathophysiology in mice fed an obesogenic Western-style diet (WD). Total Plin2 deletion (Plin2-Null) fully protected WD-fed mice from obesity, insulin resistance, adipose inflammation, steatohepatitis (NASH) and liver fibrosis found in WT animals. Hepatocyte-specific Plin2 deletion (Plin2-HepKO) largely protected against NASH and fibrosis and partially protected against steatosis in WD-fed animals, but it did not protect against obesity, insulin resistance, or adipose inflammation. Significantly, total or hepatocyte-specific Plin2 deletion impaired WD-induced monocyte recruitment and pro-inflammatory macrophage polarization found in livers of WT mice. Analyses of the molecular and cellular processes mediating steatosis, inflammation and fibrosis identified differences in total and hepatocyte-specific actions of Plin2 on the mechanisms promoting NAFLD pathophysiology. Our results demonstrate that hepatocyte-specific actions of Plin2 are central to the initiation and pathological progression of NAFLD in obese and insulin-resistant mice through effects on immune cell recruitment and fibrogenesis. Conversely, extra-hepatocyte Plin2 actions promote NAFLD pathophysiology through effects on obesity, inflammation and insulin resistance. Our findings provide new insight into hepatocyte and extra-hepatocyte mechanisms underlying NAFLD development and progression.

Different effects of global osteopontin and macrophage osteopontin in glomerular injury.

Osteopontin (OPN) is a pro-and anti-inflammatory molecule that simultaneously attenuates oxidative stress. Both inflammation and oxidative stress play a role in the pathogenesis of glomerulonephritis and in the progression of kidney injury. Importantly, OPN is highly induced in nephritic kidneys. To characterize further the role of OPN in kidney injury we used OPN-/- mice in antiglomerular basement membrane reactive serum-induced immune (NTS) nephritis, an inflammatory and progressive model of kidney disease. Normal wild-type (WT) and OPN-/- mice did not show histological differences. However, nephritic kidneys from OPN-/- mice showed severe damage compared with WT mice. Glomerular proliferation, necrotizing lesions, crescent formation, and tubulointerstitial injury were significantly higher in OPN-/- mice. Macrophage infiltration was increased in the glomeruli and interstitium in OPN-/- mice, with higher expression of IL-6, CCL2, and chemokine CXCL1. In addition, collagen (Col) I, Col III, and Col IV deposition were increased in kidneys from OPN-/- mice. Elevated expression of the reactive oxygen species-generating enzyme Nox4 and blunted expression of Nrf2, a molecule that inhibits reactive oxygen species and inflammatory pathways, was observed in nephritic kidneys from OPN-/- mice. Notably, CD11b diphteria toxin receptor mice with NTS nephritis selectively depleted of macrophages and reconstituted with OPN-/- macrophages showed less kidney injury compared with mice receiving WT macrophages. These findings suggest that in global OPN-/- mice there is increased inflammation and redox imbalance that mediate kidney damage. However, absence of macrophage OPN is protective, indicating that macrophage OPN plays a role in the induction and progression of kidney injury in NTS nephritis.

Sevelamer Improves Steatohepatitis, Inhibits Liver and Intestinal Farnesoid X Receptor (FXR), and Reverses Innate Immune Dysregulation in a Mouse Model of Non-alcoholic Fatty Liver Disease.

Bile acid sequestrants are synthetic polymers that bind bile acids in the gut and are used to treat dyslipidemia and hyperphosphatemia. Recently, these agents have been reported to lower blood glucose and increase insulin sensitivity by altering bile acid signaling pathways. In this study, we assessed the efficacy of sevelamer in treating mice with non-alcoholic fatty liver disease (NAFLD). We also analyzed how sevelamer alters inflammation and bile acid signaling in NAFLD livers. Mice were fed a low-fat or Western diet for 12 weeks followed by a diet-plus-sevelamer regimen for 2 or 12 weeks. At the end of treatment, disease severity was assessed, hepatic leukocyte populations were examined, and expression of genes involved in farnesoid X receptor (FXR) signaling in the liver and intestine was analyzed. Sevelamer treatment significantly reduced liver steatosis and lobular inflammation. Sevelamer-treated NAFLD livers had notably fewer pro-inflammatory infiltrating macrophages and a significantly greater fraction of alternatively activated Kupffer cells compared with controls. Expression of genes involved in FXR signaling in the liver and intestine was significantly altered in mice with NAFLD as well as in those treated with sevelamer. In a mouse model of NAFLD, sevelamer improved disease and counteracted innate immune cell dysregulation in the liver. This study also revealed a dysregulation of FXR signaling in the liver and intestine of NAFLD mice that was counteracted by sevelamer treatment.

Macrophage A2A Adenosine Receptors Are Essential to Protect from Progressive Kidney Injury.

A2A adenosine receptors (A2ARs) are endogenous inhibitor of inflammation. Macrophages that are key effectors of kidney disease progression express A2ARs. We investigated the role of A2ARs in kidney inflammation in a macrophage-mediated anti-glomerular basement membrane reactive serum-induced immune nephritis in A2AR-deficient mice. Sub-threshold doses of glomerular basement membrane-reactive serum induced more severe and prolonged kidney damage with higher levels of proinflammatory cytokines and greater accumulation of inflammatory cells in A2AR(-/-) mice than wild-type (WT) mice. To investigate the role of macrophage A2AR in progressive kidney injury, glomerulonephritis was induced in CD11b-DTR transgenic mice. Macrophages were selectively depleted in the established phase of the disease and reconstituted with macrophages from WT or A2AR-deficient mice and then treated with an A2AR agonist. In mice receiving WT macrophages and treated with an A2AR agonist, the glomerular cellularity, crescent formation, sclerotic glomeruli, and tubulointerstitial injury were significantly reduced compared with the control group. In contrast, in mice reconstituted with A2AR-deficient macrophages and treated with an A2AR agonist, the kidney injury was more severe with increased deposition of collagen I, III, and IV. These findings suggest that disruption of the protective A2AR amplifies inflammation to accelerate glomerular damage and endogenous macrophage A2ARs are essential to protect from progressive kidney fibrosis.

Bile acid receptor activation modulates hepatic monocyte activity and improves nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) affects a large proportion of the American population. The spectrum of disease ranges from bland steatosis without inflammation to nonalcoholic steatohepatitis and cirrhosis. Bile acids are critical regulators of hepatic lipid and glucose metabolism and signal through two major receptor pathways: farnesoid X receptor (FXR), a member of the nuclear hormone receptor superfamily, and TGR5, a G protein-coupled bile acid receptor (GPBAR1). Both FXR and TGR5 demonstrate pleiotropic functions, including immune modulation. To evaluate the effects of these pathways in NAFLD, we treated obese db/db mice with a dual FXR/TGR5 agonist (INT-767) for 6 weeks. Treatment with the agonist significantly improved the histological features of nonalcoholic steatohepatitis. Furthermore, treatment increased the proportion of intrahepatic monocytes with the anti-inflammatory Ly6C(low) phenotype and increased intrahepatic expression of genes expressed by alternatively activated macrophages, including CD206, Retnla, and Clec7a. In vitro treatment of monocytes with INT-767 led to decreased Ly6C expression and increased IL-10 production through a cAMP-dependent pathway. Our data indicate that FXR/TGR5 activation coordinates the immune phenotype of monocytes and macrophages, both in vitro and in vivo, identifying potential targeting strategies for treatment of NAFLD.

Galectin-9 functionally impairs natural killer cells in humans and mice.

Galectin-9 is a pleiotropic immune modulator affecting numerous cell types of innate and adaptive immunity. Patients with chronic infection with either hepatitis C virus (HCV) or HIV have elevated circulating levels. Limited data exist on the regulation of natural killer (NK) cell function through interaction with galectin-9. We found that galectin-9 ligation downregulates multiple immune-activating genes, including eight involved in the NK cell-mediated cytotoxicity pathway, impairs lymphokine-activated killing, and decreases the proportion of gamma interferon (IFN-γ)-producing NK cells that had been stimulated with interleukin-12 (IL-12)/IL-15. We demonstrate that the transcriptional and functional changes induced by galectin-9 are independent of Tim-3. Consistent with these results for humans, we find that the genetic absence of galectin-9 in mice is associated with greater IFN-γ production by NK cells and enhanced degranulation. We also show that in the setting of a short-term (4-day) murine cytomegalovirus infection, terminally differentiated NKs accumulate in the livers of galectin-9 knockout mice, and that hepatic NKs spontaneously produce significantly more IFN-γ in this setting. Taken together, our results indicate that galectin-9 engagement impairs the function of NK cells, including cytotoxicity and cytokine production.

High-fat and high-sucrose (western) diet induces steatohepatitis that is dependent on fructokinase.

UNLABELLED: Fructose intake from added sugars has been implicated as a cause of nonalcoholic fatty liver disease. Here we tested the hypothesis that fructose may interact with a high-fat diet to induce fatty liver, and to determine if this was dependent on a key enzyme in fructose metabolism, fructokinase. Wild-type or fructokinase knockout mice were fed a low-fat (11%), high-fat (36%), or high-fat (36%) and high-sucrose (30%) diet for 15 weeks. Both wild-type and fructokinase knockout mice developed obesity with mild hepatic steatosis and no evidence of hepatic inflammation on a high-fat diet compared to a low-fat diet. In contrast, wild-type mice fed a high-fat and high-sucrose diet developed more severe hepatic steatosis with low-grade inflammation and fibrosis, as noted by increased CD68, tumor necrosis factor alpha, monocyte chemoattractant protein-1, alpha-smooth muscle actin, and collagen I and TIMP1 expression. These changes were prevented in the fructokinase knockout mice. CONCLUSION: An additive effect of high-fat and high-sucrose diet on the development of hepatic steatosis exists. Further, the combination of sucrose with high-fat diet may induce steatohepatitis. The protection in fructokinase knockout mice suggests a key role for fructose (from sucrose) in this development of steatohepatitis. These studies emphasize the important role of fructose in the development of fatty liver and nonalcoholic steatohepatitis.

Vertical Transfer of Maternal Gut Microbes to Offspring of Western Diet-Fed Dams Drives Reduced Levels of Tryptophan Metabolites and Postnatal Innate Immune Response.

Maternal obesity and/or Western diet (WD) is associated with an increased risk of metabolic dysfunction-associated steatotic liver disease (MASLD) in offspring, driven, in part, by the dysregulation of the early life microbiome. Here, using a mouse model of WD-induced maternal obesity, we demonstrate that exposure to a disordered microbiome from WD-fed dams suppressed circulating levels of endogenous ligands of the aryl hydrocarbon receptor (AHR; indole, indole-3-acetate) and TMAO (a product of AHR-mediated transcription), as well as hepatic expression of Il10 (an AHR target), in offspring at 3 weeks of age. This signature was recapitulated by fecal microbial transfer from WD-fed pregnant dams to chow-fed germ-free (GF) lactating dams following parturition and was associated with a reduced abundance of Lactobacillus in GF offspring. Further, the expression of Il10 was downregulated in liver myeloid cells and in LPS-stimulated bone marrow-derived macrophages (BMDM) in adult offspring, suggestive of a hypo-responsive, or tolerant, innate immune response. BMDMs from adult mice lacking AHR in macrophages exhibited a similar tolerogenic response, including diminished expression of Il10. Overall, our study shows that exposure to maternal WD alters microbial metabolites in the offspring that affect AHR signaling, potentially contributing to innate immune hypo-responsiveness and progression of MASLD, highlighting the impact of early life gut dysbiosis on offspring metabolism. Further investigations are warranted to elucidate the complex interplay between maternal diet, gut microbial function, and the development of neonatal innate immune tolerance and potential therapeutic interventions targeting these pathways.

Advanced age worsens respiratory function and pulmonary inflammation after burn injury and this correlates with changes in the fecal microbiome in mice.

Cutaneous burn injury in the elderly is associated with poor clinical outcomes and increased pulmonary-related complications. We and others have shown that burn injury triggers a cascade of inflammatory mediators which increase gut permeability and dysbiosis of the fecal microbiota and this is more dramatic in the aged. Since cross-talk between intestinal microbes and the lung, termed the "gut-lung axis," impacts immunity and homeostasis in the airway, we hypothesized that the increased intestinal dysbiosis in age and burn injury may contribute to excessive pulmonary inflammation and poor prognosis after injury. To explore this hypothesis, we used a clinically relevant murine model of burn injury in which young and aged mice are subjected to a 12% total body surface area dorsal scald burn or sham injury. 24 hours after injury, lung function was assessed and lungs and feces were collected for analysis of inflammatory mediators and fecal microbial species. The results show that, when compared to younger mice, burn injury in aged mice triggers a decline in respiratory function and exacerbates pulmonary inflammation. In addition to heightened levels of the neutrophil recruiting chemokine CXCL1, aged mice displayed a profound increase in the pro-inflammatory protein, calprotectin, in the lung after burn injury. Comparison of the fecal microbiome and inflammatory markers in the lung revealed unique, age-dependent, correlation patterns between individual taxa and pulmonary inflammation. Taken together, these findings suggest that the post-burn dysbiosis of the gut flora in aged mice may contribute to the changes in pulmonary inflammatory profiles.

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.

Peptide vaccines prevent tumor growth by activating T cells that respond to native tumor antigens.

Peptide vaccines enhance the response of T cells toward tumor antigens and represent a strategy to augment antigen-independent immunotherapies of cancer. However, peptide vaccines that include native tumor antigens rarely prevent tumor growth. We have assembled a set of peptide variants for a mouse-colon tumor model to determine how to improve T-cell responses. These peptides have similar affinity for MHC molecules, but differ in the affinity of the peptide-MHC/T-cell receptor interaction with a tumor-specific T-cell clone. We systematically demonstrated that effective antitumor responses are generated after vaccination with variant peptides that stimulate the largest proportion of endogenous T cells specific for the native tumor antigen. Importantly, we found some variant peptides that strongly stimulated a specific T-cell clone in vitro, but elicited fewer tumor-specific T cells in vivo, and were not protective. The T cells expanded by the effective vaccines responded to the wild-type antigen by making cytokines and killing target cells, whereas most of the T cells expanded by the ineffective vaccines only responded to the peptide variants. We conclude that peptide-variant vaccines are most effective when the peptides react with a large responsive part of the tumor-specific T-cell repertoire.

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.

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.