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Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish

ABSTRACT: Neutrophils accumulate early in burn wounds, and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However, we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here, we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single-cell transcriptional (scRNA-Seq) mapping of myeloid cells during a 3-day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions, we found subtype-specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils, with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality, suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore, we characterized the transcriptional dynamics of cell-cell interactions in both conditions by time. Among burn-induced signaling pathways, we found il6-il6r signaling as a time-point-specific macrophage-neutrophil interaction, suggesting the importance of timing in innate immune response in burn. Finally, to test the translational value of our fish model, we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area (TBSA) in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single-cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients.

ORGANISM(S): Danio rerio

PROVIDER: GSE250610 | GEO | 2024/07/26

REPOSITORIES: GEO

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Project description:Thermal injury incites inflammatory responses that often transcend the local environment and lead to structural deficiencies in skin that give way to scar formation. We hypothesized that extensive perturbations within burned skin following thermal insult and during subsequent events of wound repair induce vast alterations in gene expression that likely serve as a wound and systemic healing deterrent. A high-throughput microarray experiment was designed to analyze genetic expression patterns and identify potential genes to target for therapeutic augmentation or silencing. The study compares gene expression from burn wound margins at various times following thermal injury to expression observed in normal skin. Utilizing this design, we report that the totality of gene expression alterations is indeed enormous. Further, we observed that the differential expression of many inflammatory and immune response genes appear to be continually up-regulated in burn wound margins seven days or more after initial thermal insult. As it is well established that the inflammatory process must abate for wound healing to proceed, the finding of ongoing local inflammation is cause for further investigation. To our knowledge, this is the first report of the gene expression alterations induced by thermal injury of human skin. As such, it provides a wealth of data to mine with the ultimate goal of better understanding the local pathophysiologic changes at the site of thermal injury that not only affect wound healing capacity, but may also contribute to systemic derangements within the burn patient. Keywords: time course, disease state analysis The study compares gene expression from burn wound margins at various times following thermal injury to expression observed in normal skin. All skin specimens were obtained in the operating room within minutes of being removed from the patient. Burn specimens were taken from wound margins. Harvested tissue at the burn wound margin maximized the capture of viable cells from the multiple lineages important to the healing process and minimized the inclusion of non-viable cells destroyed by full-thickness injury. After isolation, RNA samples were pooled equally by mass as to contain RNA from 5 tissue specimens for each array replicate.

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Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish

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