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Time-resolved single-cell RNAseq profiling identifies a novel Fabp5-expressing subpopulation of inflammatory myeloid cells in chronic spinal cord injury

ABSTRACT: Traumatic spinal cord injury (SCI) is a debilitating central nervous system (CNS) pathology that lacks effective restorative treatments. The mechanisms of tissue recovery after SCI are dysregulated in comparison to normal wound healing, leading to a chronic wound state, rather than a return to homeostasis. While this state is characterized by persistent inflammation driven by CNS resident microglia (MG) and infiltrating myeloid cells, the precise roles of myeloid cell subsets after SCI remains unclear. In particular, upon crossing the blood-brain barrier, tissue infiltrating monocyte-derived macrophages (MCd) take on the morphology of MG, and upregulate canonical MG markers2, making the two populations practically indistinguishable. Here, we first employed a Cx3cr1-CreERT2 mouse model to label Cx3cr1+ cells for FACS isolation. Then we utilized fate-mapping approach (see methods) to investigate the ontology and temporal dynamics of Cx3cr1+ spinal cord MG (RFP+/YFP+) and Cx3cr1+ infiltrating myeloid cells (RFP-/YFP+) via FACS isolation and single-cell RNA sequencing (scRNAseq) in a mouse model of thoracic contusion SCI. Useful Links: Github repository (https://github.com/regan-hamel/SCI_2020)

ORGANISM(S): Mus musculus

PROVIDER: GSE159638 | GEO | 2020/10/23

REPOSITORIES: GEO

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Project description:The inflammatory response after spinal cord injury (SCI) is an important contributor to secondary damage. Infiltrating macrophages can acquire a spectrum of activation states, however, the microenvironment at the SCI site favors macrophage polarization into a pro-inflammatory phenotype, which is one of the reasons why macrophage transplantation has failed. In this study, we investigated the therapeutic potential of the macrophage secretome for SCI recovery. We investigated the effect of the secretome in vitro using peripheral and CNS-derived neurons and human neural stem cells. Moreover, we perform a pre-clinical trial using a SCI compression mice model and analyzed the recovery of motor, sensory and autonomic functions. Instead of transplanting the cells, we injected the paracrine factors and extracellular vesicles that they secrete, avoiding the loss of the phenotype of the transplanted cells due to local environmental cues. We demonstrated that different macrophage phenotypes have a distinct effect on neuronal growth and survival, namely, the alternative activation with IL-10 and TGF-β1 (M(IL-10+TGF-β1)) promotes significant axonal regeneration. We also observed that systemic injection of soluble factors and extracellular vesicles derived from M(IL-10+TGF-β1) macrophages promotes significant functional recovery after compressive SCI and leads to higher survival of spinal cord neurons. Additionally, the M(IL-10+TGF-β1) secretome supported the recovery of bladder function and decreased microglial activation, astrogliosis and fibrotic scar in the spinal cord. Proteomic analysis of the M(IL-10+TGF-β1)-derived secretome identified clusters of proteins involved in axon extension, dendritic spine maintenance, cell polarity establishment, and regulation of astrocytic activation. Overall, our results demonstrated that macrophages-derived soluble factors and extracellular vesicles might be a promising therapy for SCI with possible clinical applications.

Project description:Purpose: Spinal cord injury (SCI) is a devastating neurological disease without effective treatment. To generate a comprehensive view of the mechanisms involved in SCI pathology, we applied RNA-sequencing (RNA-seq) technology to characterize the temporal changes in global gene expression after contusive SCI in mice. Method Part1: A total of 27 female C57BI/6J mice (10-16 weeks of age; 20-25g; The Jackson Laboratory, Bar Harbor, ME) were used with 9 mice in each of following groups: shame control, 2 and 7 days after SCI. The surgical procedure for SCI were described previously [PMID:23289019]. Briefly, after anesthetization with a mixed solution of ketamine (80 mg/kg, ip) and xylazine (10 mg/kg, ip), mice received a dorsal laminectomy at the 9th thoracic vertebral (T9) level to expose the spinal cord and then a moderate T9 contusive injury using an Infinite Horizons impactor (PrecisionSystems and Instrumentation) at 60 kdyn with the spinestabilized using steel stabilizers inserted under the transverse processes one vertebra above and below the injury [PMID:19196178] . The shame control mice received only a dorsal laminectomy without contusive injury. Afterwards, the wound was sutured in layers, bacitracin ointment(Qualitest Pharmaceuticals,Huntsville, AL) was applied tothe wound area, 0.1mL of a 20 mg/ml stock of gentamicin(ButlerSchein, Dublin, OH) was injected subcutaneously, and the animals recovered on a water-circulating heating pad. Then mice received analgesic agent, buprenorphin(0.05 mg/kg, SQ; Reckitt Benckise, Hull, England)twice a day for two days. Bladders were manually expressed until automatic voiding returned spontaneously, which generally was within 7 days. At 2 or 7 days after SCI, the mice were anesthetized again with ketamine and xylazine and perfused briefly with normal physical saline. The injured spinal cords were then dissected and three 0.5 mm pieces of spinal cord were cut in the injured epicenter. All spinal cords were immediately frozen in liquid nitrogen and processed for RNA isolation. The spinal cords from three mice were combined into one biological replicate for RNA extraction. Three biological replicates were used. Method Part2: RNA-Seq was performed on the polyadenylated fraction of RNA isolated from tissue samples of acute (2D) and subacute phase (7D) and normal tissues (control, denoted as CTR hereafter). Three biological replicates were used for each phase.150-300 ng total RNAs were used for each sequencing library. RNA samples were polyA selected and paired-end sequencing libraries were constructed using TruSeq RNA Sample Prep Kit as described in the TruSeq RNA Sample Preparation V2 Guide (Illumina).The samples were then sequenced using the Illumina HiSeq sequencer. More than 30 million 100bp paired-end reads were generated from each biological replicate. Method Part3: Read mapping and Transcriptome construction were done by using optimized pipeline which integrate Tophat followed by Cufflinks. Result: We sequenced tissue samples from acute and subacute phases (2 days and 7 days after injury) and systematically characterized the transcriptomes with the goal of identifying pathways and genes critical in SCI pathology. The top enriched functional categories include ‘inflammation response’, ‘neurological disease’, ‘cell death and survival’ and ‘nervous system development’. The top enriched pathways include LXR/RXR Activation and Atherosclerosis Signaling etc. Furthermore, we developed a systems-based analysis framework in order to identify key determinants in the global gene networks of the acute and sub-acute phases. Some candidate genes that we identified have been shown to play important roles in SCI, which demonstrates the validity of our approach. There are also many genes whose functions in SCI have not been well studied and can be further investigated by future experiments. We have also incorporated pharmacogenomic information into our analyses. Among the genes identified, the ones with existing drug information can be readily tested in SCI animal models. Conclusion: in this study we have described an example of how global gene profiling can be translated to screening genes of interest and generating new hypotheses. Additionally, the RNA-seq enables splicing isoform identification and the estimation of expression levels, thus providing useful information for increasing the specificity of drug design and reducing potential side effect. In summary, these results provide a valuable reference data resource for a better understanding of the SCI process in the acute and sub-acute phases. mRNA profiles of Acute/subacute phase Spinal Cord Injury sample from mice were generated by RNA-sequencing using Illumina HiSeq.

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Time-resolved single-cell RNAseq profiling identifies a novel Fabp5-expressing subpopulation of inflammatory myeloid cells in chronic spinal cord injury

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