Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish
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PROVIDER: PRJNA1054617 | ENA |
REPOSITORIES: ENA
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Project description: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.
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Project description:Burn wound blister fluid is a valuable matrix for understanding the biological pathways associated with burn injury. In this study, 152 blister fluid samples collected from paediatric burn wounds at two different hospitals were analysed using mass spectrometry proteomic techniques. The protein abundance profile at different days post-burn indicated that there were more proteins associated with cellular damage/repair in the first 24 hours, whereas after this point there were more proteins associated with antimicrobial defence and inflammation. The inflammatory proteins persisted at a high level in the blister fluid for more than 7 days. This may indicate that removal of burn blisters prior to two days post-burn is optimal to prevent excessive or prolonged inflammation in the wound environment. Additionally, many proteins associated with the neutrophil extracellular trap (NET) pathway were increased post-burn, further implicating NETs in the post-burn inflammatory response. NET inhibitors may therefore be a potential treatment to reduce post-burn inflammation and coagulation pathology and enhance burn wound healing outcomes.
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Project description:Infections of burn wounds, especially those caused by Pseudomonas aeruginosa, could trigger sepsis or septic shock, which is the main cause of death after burn injury. Compared with traditional saline-wet-to-dry dressings, negative pressure wound therapy (NPWT) is more effective for the prevention and treatment of wound infections. However, the mechanism by which NPWT controls infection and accelerates wound healing remains unclear. Accordingly, in this study, the molecular mechanisms underlying the effects of NPWT were explored using a murine model of P. aeruginosa-infected burn wounds. NPWT significantly reduced P. aeruginosa levels in wounds, enhanced blood flow, and promoted wound healing. Additionally, NPWT markedly alleviated wound inflammation and increased the expression of wound healing–related molecules. Recent evidence points to a role of circular RNAs (circRNAs) in wound healing; hence, whole-transcriptome sequencing of wound tissues from NPWT and control groups was performed to evaluate circRNA expression profiles.
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Project description:#G017 Burn/Trauma Study. This is a Glue Grant Study comparison between gene expression analyses performed in eight trauma/burn subjects using either a buffy coat isolation of whole blood or using the PAXgene system Keywords: other
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Project description:Animal experiments were performed with male Sprague-Dawley rats (Charles River Laboratories, Boston, MA) weighing 150 ? 200 grams. All experimental protocols used in this study were approved by the Subcommittee on Research Animal Care, Massachusetts General Hospital. Rats were individually housed in a temperature-controlled (25oC) and light-controlled room (12h light-dark cycle) and allowed to adjust to their new surroundings for at least 5 days prior to the experiment. Water and rat chow were provided ad libitum to the animals. On the day of the treatment, the animals were randomly divided into two groups, burned and sham-burned. The burn injury consisted of a full-skin-thickness scald burn of the dorsum, calculated to be ~ 20% of the rat?s total body surface area (TBSA), induced by immersing the designated area in boiling water for 10 s (45). Rats were resuscitated with an intra-peritoneal injection of sterile saline solution (1.5 mL/Kg body weight/% TBSA) immediately after burn. The mortality rate of this treatment was negligible. At each time point (1, 2, 4, and 7d), three animals belonging to each group were sacrificed, the liver and serum collected, and stored at ?80oC after being inflicted with 20% TBSA burn injury. Sham-burn rats (n=3) considered as the control were treated identically except that they were immersed into a 37oC water bath and immediately sacrificed to collect the livers. Keywords = Burn Liver microarray genechip time course Keywords: time-course
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Project description:Pseudomonas aeruginosa strain:burn wound | isolate:burn wound Genome sequencing
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Project description:Burn injury remains a major clinical challenge to both survival and to quality of life. Its progressive, aberrant inflammation underlies the lethal dysfunction of various organs and the pain it induces is excruciating and notoriously difficult to manage. While it is known that burn injury’s complex local and disseminating pathology is orchestrated from the burned tissue, few studies have sought to characterise the local signalling environment. An enhanced understanding of the local and acutely temporally-dynamic processes defining burn injury and its progression is required for the development of novel therapeutic interventions. Microdialysis was used as an interstitial sampling technique, conducted over three hours post-burn. Samples were analysed by metabolomics and a multiplex cytokine immunoassay. Next-Generation sequencing libraries of the burn and control microdialysis sites were prepared to measure transcriptional changes potentially underlying the interstitial profile characterising burn injury.
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Project description:scRNA and snATAC sequencing of cells harvested from the tendon injury site after a severe burn/tenotomy injury in Hoxa11 lineage traced mice allowed for differentation tracing of MSCs located in zeugopod after severe heterotopic ossification inducing injury. The use of immobilization also allowed us to determine the effects of limb immoblization on MSCs during aberrant wound healing.
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