Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish

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. Overall design: At 6/24/48 hour-post-burn (hpb), burned and unwounded larvae at matching developmental stages (3/4/5 day-post-fertilization (dpf)) were transferred to 35 mm dish containing calcium-free PBS for 15 min and then anesthetized with tricaine methanesulfonate (MS222, 200 mg/L). A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution (0.25% trypsin, 1mM EDTA in PBS) at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution (1mM CaCl2, 100% FBS). Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3,000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI (1mg/mL). Fluorescence activated cell sorting (FACS) was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris, singles versus doublets, live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ (488 nm channel) and mCherry+ (561 nm channel) cells were sorted into PBS with 10% FBS. Post sorting, cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin-Madison Biotechnology Center (RRID: SCR_017757). Samples were processed with Chromium Single Cell Gene Expression Solution 3' v2 (10X Genomics).