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.

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.

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.

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.

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.

Longitudinal microbiome analysis of single donor fecal microbiota transplantation in patients with recurrent Clostridium difficile infection and/or ulcerative colitis.

BACKGROUND: Studies of colonoscopic fecal microbiota transplant (FMT) in patients with recurrent CDI, indicate that this is a very effective treatment for preventing further relapses. In order to provide this service at Stony Brook University Hospital, we initiated an open-label prospective study of single colonoscopic FMT among patients with ≥ 2 recurrences of CDI, with the intention of monitoring microbial composition in the recipient before and after FMT, as compared with their respective donor. We also initiated a concurrent open label prospective trial of single colonoscopic FMT of patients with ulcerative colitis (UC) not responsive to therapy, after obtaining an IND permit (IND 15642). To characterize how FMT alters the fecal microbiota in patients with recurrent Clostridia difficile infections (CDI) and/or UC, we report the results of a pilot microbiome analysis of 11 recipients with a history of 2 or more recurrences of C. difficile infections without inflammatory bowel disease (CDI-only), 3 UC recipients with recurrent C. difficile infections (CDI + UC), and 5 UC recipients without a history of C. difficile infections (UC-only). METHOD: V3V4 Illumina 16S ribosomal RNA (rRNA) gene sequencing was performed on the pre-FMT, 1-week post-FMT, and 3-months post-FMT recipient fecal samples along with those collected from the healthy donors. Fitted linear mixed models were used to examine the effects of Group (CDI-only, CDI + UC, UC-only), timing of FMT (Donor, pre-FMT, 1-week post-FMT, 3-months post-FMT) and first order Group*FMT interactions on the diversity and composition of fecal microbiota. Pairwise comparisons were then carried out on the recipient vs. donor and between the pre-FMT, 1-week post-FMT, and 3-months post-FMT recipient samples within each group. RESULTS: Significant effects of FMT on overall microbiota composition (e.g., beta diversity) were observed for the CDI-only and CDI + UC groups. Marked decreases in the relative abundances of the strictly anaerobic Bacteroidetes phylum, and two Firmicutes sub-phyla associated with butyrate production (Ruminococcaceae and Lachnospiraceae) were observed between the CDI-only and CDI + UC recipient groups. There were corresponding increases in the microaerophilic Proteobacteria phylum and the Firmicutes/Bacilli group in the CDI-only and CDI + UC recipient groups. At a more granular level, significant effects of FMT were observed for 81 genus-level operational taxonomic units (OTUs) in at least one of the three recipient groups (p<0.00016 with Bonferroni correction). Pairwise comparisons of the estimated pre-FMT recipient/donor relative abundance ratios identified 6 Gammaproteobacteria OTUs, including the Escherichia-Shigella genus, and 2 Fusobacteria OTUs with significantly increased relative abundance in the pre-FMT samples of all three recipient groups (FDR < 0.05), however the magnitude of the fold change was much larger in the CDI-only and CDI + UC recipients than in the UC-only recipients. Depletion of butyrate producing OTUs, such as Faecalibacterium, in the CDI-only and CDI + UC recipients, were restored after FMT. CONCLUSION: The results from this pilot study suggest that the microbial imbalances in the CDI + UC recipients more closely resemble those of the CDI-only recipients than the UC-only recipients.

Altered Interactions between the Gut Microbiome and Colonic Mucosa Precede Polyposis in APCMin/+ Mice.

Mutation of the adenomatous polyposis coli (APC gene), an early event in the adenoma-carcinoma sequence, is present in 70-80% of sporadic human colorectal adenomas and carcinomas. To test the hypothesis that mutation of the APC gene alters microbial interactions with host intestinal mucosa prior to the development of polyposis, culture-independent methods (targeted qPCR assays and Illumina sequencing of the 16S rRNA gene V1V2 hypervariable region) were used to compare the intestinal microbial composition of 30 six-week old C57BL/6 APCMin/+ and 30 congenic wild type (WT) mice. The results demonstrate that similar to 12-14 week old APCMin/+ mice with intestinal neoplasia, 6 week old APCMin/+ mice with no detectable neoplasia, exhibit an increased relative abundance of Bacteroidetes spp in the colon. Parallel mouse RNA sequence analysis, conducted on a subset of proximal colonic RNA samples (6 APCMin/+, 6 WT) revealed 130 differentially expressed genes (DEGs, fold change ≥ 2, FDR <0.05). Hierarchical clustering of the DEGs was carried out by using 1-r dissimilarity measurement, where r stands for the Pearson correlation, and Ward minimum variance linkage, in order to reduce the number of input variables. When the cluster centroids (medians) were included along with APC genotype as input variables in a negative binomial (NB) regression model, four of seven mouse gene clusters, in addition to APC genotype, were significantly associated with the increased relative abundance of Bacteroidetes spp. Three of the four clusters include several downregulated genes encoding immunoglobulin variable regions and non-protein coding RNAs. These results support the concept that mutation of the APC gene alters colonic-microbial interactions prior to polyposis. It remains to be determined whether interventions directed at ameliorating dysbiosis in APCMin/+mice, such as through probiotics, prebiotics or antibiotics, could reduce tumor formation.