Although morphologic progression coupled with expression of specific molecular markers has been characterized along the esophageal squamous differentiation gradient, the molecular heterogeneity within cell types along this trajectory has yet to be classified at the single cell level. To address this knowledge gap, we perform single cell RNA-sequencing of 44,679 murine esophageal epithelial, to identify 11 distinct cell populations as well as pathways alterations along the basal-superficial axis and in each individual population. We evaluate the impact of aging upon esophageal epithelial cell populations and demonstrate age-associated mitochondrial dysfunction. We compare single cell transcriptomic profiles in 3D murine organoids and human esophageal biopsies with that of murine esophageal epithelium. Finally, we employ pseudotemporal trajectory analysis to develop a working model of cell fate determination in murine esophageal epithelium. These studies provide comprehensive molecular perspective on the cellular heterogeneity of murine esophageal epithelium in the context of homeostasis and aging.

Background: Patients intoxicated at the time of burn experience increased rates of sepsis and death compared with that observed in similarly sized burns alone. We sought to characterise changes in the intestinal microbiome and short-chain fatty acids (SCFAs) following alcohol intoxication and burn injury and to determine whether these changes are associated with intestinal inflammation. Methods: 10–12-week-old C57BL/6 male and female mice were subjected to ethanol intoxication and a 12.5% total body surface area scald burn injury. The following day, mice were euthanised and faecal contents from the caecum and small intestine (SI) were harvested for 16S sequencing for microbial analysis and caecum contents underwent high-performance liquid chromatography mass spectroscopy to assess SCFAs. Results: The intestinal microbiome of ethanol burn (EB) mice exhibited decreased alpha diversity and distinct beta diversity compared with sham vehicle (SV). EB faeces were marked by increased Proteobacteria and many pathobionts. EB caecum faeces exhibited a significant decrease in butyrate and a downward trend in acetate and total SCFAs. SCFA changes correlated with microbial changes particularly in the SI. Treatment of murine duodenal cell clone-K (MODE-K) cells with faecal slurries led to upregulation of interleukin-6 (IL-6) from EB faeces compared with SV faeces which correlated with levels of Enterobacteriaceae. However, supplementation of butyrate reduced faecal slurry-induced MODE-K cells IL-6 release. Conclusion: Together, these findings suggest that alcohol and burn injury induce bacterial dysbiosis and a decrease in SCFAs, which together can promote intestinal inflammation and barrier disruption, predisposing to postinjury pathology.

Background: Pneumonia contributes to post-burn morbidity and mortality. Understanding the mechanisms that predispose burn patients to pneumonia is crucial to both stratifying patients at increased risk and developing targeted interventions. Methods: A prospective observational study was conducted with 47 human patients who sustained large burn injuries with serum collected on days 2 and 3 post-burn and assessed for Angiopoietin-1 (Ang-1) and -2 (Ang-2). C57BL/6 mice were subjected to either sham injury or a 12.5% total body surface area (TBSA) scald burn injury, and plasma and lungs were assessed. Results: Patients who developed pneumonia within 30 days of injury had higher serum Ang-2 and Ang-2/1 ratio on post-injury days 2 and 3. Similar to patient findings, we observed an increase in Ang-2 in burn mice compared to sham. Within the lungs of burn mice, we found significant increases in Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (TIE2) receptor transcript Tek, downstream mediators TNFAIP3 Interacting Protein 2 (Tnip2) and phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1), in addition to endothelial adhesion molecules intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), along with neutrophil infiltration and markers compared to sham. Conclusions: These findings suggest that burn injury increases Angiopoetin-2 and downstream signaling in the lungs, which may contribute to post-burn pulmonary dysfunction. Further studies are necessary to understand if modulating the Ang–TIE2 axis can protect against pneumonia post-burn.

Background: Alcohol consumption has been found to worsen the pathology of colitis. To elucidate the underlying mechanism, we performed both miRNA and mRNA sequencing of the large intestinal epithelial cells derived from a murine model of alcohol‐induced colitis. Methods: Mice received Dextran Sulfate Sodium (DSS) for 5 days to induce colitis and were gavaged with alcohol (~3 g/kg) or water for 3 days. Three hours after the final gavage, mice were euthanized, and colon lengths were measured, followed by epithelial cell isolation. RNA, including miRNA, was isolated from the colonic epithelial cells and sent to Novogene for mRNA and miRNA sequencing. Results: Analysis comparing DSS Ethanol vs. Sham Vehicle mice showed 3931 significantly upregulated genes and 3788 significantly downregulated genes. Functional enrichment analysis of GO and KEGG pathways showed enrichment of positive regulation of superoxide anion generation and HIF‐1 signaling pathways, both related to oxidative stress. DSS Ethanol compared to DSS Vehicle resulted in 921 significantly upregulated and 1072 significantly downregulated genes ( P _adj < 0.05), with functional enrichment analysis showing the term with the highest fold enrichment was superoxide anion generation. We further confirmed the RNA expression of NOS2, CYBB, and NCF1, which are involved in nitric oxide and superoxide production. miRNA sequencing revealed 84 differentially expressed miRNAs comparing DSS Ethanol to DSS Vehicle. Functional enrichment analysis of predicted targets of these miRNAs also showed enrichment in superoxide anion generation. Conclusion: These results suggest that combined ethanol and DSS exposure may increase the production of reactive oxygen species, which further damages the intestinal lining and potentially contributes to the ethanol exacerbation of colitis.

Background: Severe burn injury induces gastrointestinal dysfunction and bacterial dysbiosis, yet the systemic impact of altered bacterial metabolites remains poorly understood. We assessed whether changes in plasma levels of trimethylamine N-oxide (TMAO), a gut-derived metabolite, have a relationship with changes in the whole blood transcriptomic profile following burn injury. Methods: Using previously harvested plasma from 67 burn patients, we quantified circulatory levels of TMAO via ELISA. To assess TMAO’s impact on the blood transcriptome, we applied a linear model to previously published whole blood microarray data from a subset of these patients (n = 22) with paired TMAO measurements. Results: While initial TMAO levels were comparable across small and larger burns, concentrations significantly increased 24-36 h post-admission in patients with >10% total body surface area (TBSA) burns, independent of age or sex. Elevated TMAO positively correlated with both burn size and Baux score. Furthermore, transcriptomic analysis revealed that high TMAO levels were strongly associated with the enrichment of inflammatory and leukocyte activation pathways, despite a lack of association between TMAO levels and imputed fractions of immune cells. Conclusion: Burn injury is associated with a delayed, severity-dependent elevation in circulating TMAO. This increase in TMAO correlates with transcriptomic signatures of robust immune activation and systemic inflammation, suggesting that targeting the gut microbiome and its metabolites may offer a novel therapeutic avenue to address post-burn inflammatory complications.