RNA-seq of Tregs from injured mouse neonatal and adult tendon and spleen.
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ABSTRACT:
PROVIDER: PRJNA726990 | ENA |
REPOSITORIES: ENA
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Project description:We report transcriptomic differences between neonatal and adult Treg responses to tendon jury using NGS sequencing. We performed tendon injuries to neonatal mice at P5 and 4-6 month old adult mice and isolated Tregs by FACS (CD4-PE+, Foxp3-GFP+) from injured tendons and autologus spleens 14 days after inury. We then performed RNA-sequencing with biological triplicates to distinguish unique transcriptomic signatures that can illuminate the role of Tregs in neonatal tendon regeneration. Tregs from neonatal tendon show a type 2 immune signatures with a unique gene signature that is distinc from canonical Treg activation, while adult Tregs demonstrate a type 1 signature.
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Project description:We aimed to identify the changes in gene expression profile of Tregs isolated from the injured bone, muscle, skin and heart, compared to healthy (uninjured) spleen Tregs. The bone injury consisted of calvarial bone defects, the muscle injury involved volumetric muscle loss defect in the quadriceps and the skin injury involved full-thickness punch-biopsy wounds. The heart injury was performed using the left coronary artery ligation to induce myocardial infarction (MI). The Tregs accumulating within the injured tissues were isolated and purified by fluorescence activated cell sorting (FACS), at 7 days post-injury (which is the peak of Treg accumulation within these tissues post-injury).
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Project description:Transcriptomic profile of recovered exogenous Tregs from injured bone, muscle, and skin of mice that were locally treated with Tregs, as well as heart, mediastinal lymph nodes (MLN) and spleen from mice with myocardial infarcts (MI) that were systemically treated with Tregs. Mice with tissue injuries were treated with exogenous Tregs that were sorted from spleens of Foxp3(IRES-mRFP) mice - C57BL6/J mice with bone, muscle and skin injuries were treated via hydrogel-mediated local delivery of Tregs soon after injury, while mice with myocardial infarcts (MI) were treated with systemic (intravenous) delivery of Tregs one day post-MI. Three days after Treg-delivery, the injured bone, muscle, and skin tissues, as well as heart, mediastinal lymph nodes and spleens from mice with MI were harvested, and the exogenous (delivered) Tregs were recovered by FACS sorting. These sorted exogenous Tregs recovered at day 3 post-Treg delivery (Day 3 recovered Tregs) were then used for mini-bulk RNA sequencing along with spleen Tregs before delivery as a control (Day 0 Spleen Tregs).
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Project description:A phenotypically and functionally distinct population of CD4+ Foxp3+ T cells (Tregs) rapidly accumulates in acutely injured skeletal muscle of mice, just as invading myeloid-lineage cells switch from a pro-inflammatory to a pro-regenerative state. Analysis of gene expression of Tregs and CD4+Foxp3- T cells (Tconvs) from injured muscle and spleen revealed that the transcriptome of muscle Treg cells is distinct from that of splenic Tregs. A set of genes is uniquely expressed by muscle Tregs, while another set is over-expressed by the two muscle populations vis-à-vis their two spleen counterparts. 6 wk-old Foxp3-ires-GFP mice were injured in skeletal muscles with cardiotoxin. Four and fourteen days later, Tregs and Tconvs from spleen and muscle were double-sorted into Trizol. To reduce variability, cells from multiple mice were pooled for sorting, and three replicates were generated for all groups. RNA from 1.5-2.5 x 104 cells was amplified, labeled, and hybridized to Affymetrix Mouse Gene 1.0 ST Arrays.
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Project description:Transcriptomic profile of endogenous Tregs in mouse injured bone, muscle, skin and heart tissues compared to healthy spleen Tregs
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Project description:A phenotypically and functionally distinct population of CD4+ Foxp3+ T cells (Tregs) rapidly accumulates in acutely injured skeletal muscle of mice, just as invading myeloid-lineage cells switch from a pro-inflammatory to a pro-regenerative state. Analysis of gene expression of Tregs and CD4+Foxp3- T cells (Tconvs) from injured muscle and spleen revealed that the transcriptome of muscle Treg cells is distinct from that of splenic Tregs. A set of genes is uniquely expressed by muscle Tregs, while another set is over-expressed by the two muscle populations vis-à-vis their two spleen counterparts.
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Project description:Rotator cuff injuries result in over 500,000 surgeries performed annually, an alarmingly high number of which fail. These procedures typically involve repair of the injured tendon and removal of the subacromial bursa. However, recent identification of a resident population of mesenchymal stem cells and inflammatory responsiveness of the bursa to tendinopathy indicate an unexplored biological role of the bursa in the context of rotator cuff disease. Therefore, we aimed to understand the clinical relevance of bursa-tendon crosstalk, characterize the biologic role of the bursa within the shoulder, and test the therapeutic potential for targeting the bursa. Proteomic profiling of patient bursa and tendon samples demonstrated that the bursa is activated by tendon injury. Using a rat to model rotator cuff injury and repair, tenotomy-activated bursa protected the intact tendon adjacent to the injured tendon and maintained the morphology of the underlying bone. The bursa also promoted an early inflammatory response in the injured tendon, initiating key players in wound healing. In vivo results were supported by targeted organ culture studies of the bursa. To examine the potential to therapeutically target the bursa, dexamethasone was delivered to the bursa, prompting a shift in cellular signaling towards modulating inflammation in the healing tendon. In conclusion, contrary to current clinical practice, the bursa should be retained to the greatest extent possible and provides a new therapeutically target for improving tendon healing outcomes.
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Project description:We report age-related gene expression of Treg cells isolated from injured muscle and spleen. Male C57BL/6 Foxp3-GFP reporter mice were injured intramuscularly with cardiotoxin. Tregs were sorted directly into Trizol from injured muscle and spleen 4 days post-injury. Gene expression profiling of muscle and splenic Tregs from 2- vs >6-month old mice (biological duplicate for each).
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