Abstract
Fibrosis is a common pathological response to inflammation in many peripheral tissues and can prevent tissue regeneration and repair. Here, we identified persistent fibrotic scarring in the CNS following immune cell infiltration in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Using lineage tracing and single-cell sequencing in EAE, we determined that the majority of the fibrotic scar is derived from proliferative CNS fibroblasts, not pericytes or infiltrating bone marrow-derived cells. Ablating proliferating fibrotic cells using cell-specific expression of herpes thymidine kinase led to an increase in oligodendrocyte lineage cells within the inflammatory lesions and a reduction in motor disability. We further identified that interferon-gamma pathway genes are enriched in CNS fibrotic cells, and the fibrotic cell-specific deletion of Ifngr1 resulted in reduced fibrotic scarring in EAE. These data delineate a framework for understanding the CNS fibrotic response.
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Data availability
All RNA-seq and single-cell sequencing raw and processed data files have been uploaded to the Gene Expression Omnibus and can be accessed using the following accession codes: GSE135186 for single-cell transcriptional profile of Col1a1-GFP+ cells in healthy and EAE samples; GSE135044 for transcriptional profile of Col1a1-GFP+ CNS cells in healthy and EAE samples; and GSE135185 for single-cell transcriptional profile of a mouse spinal cord in EAE.
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Acknowledgements
We thank K. Jepsen and the UCSD Institute of Genomic Medicine Genomics center, T. Lambardo and the UCSD Veterans Hospital Flow Cytometry Core, Y. Jones and the CMM UCSD Electron Microscopy Core and J. Santini and M. Erb at the UCSD Microscopy Core. We thank D. Brenner (UCSD Health Sciences) for kindly supplying the Col1a1-GFP mice. R.D. is funded by National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) awards (R01 NS091281, R01 NS103844), a National Multiple Sclerosis Society grant and the UCSF Program for Breakthrough Biomedical Science. C.E.D. is funded by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences (T32 GM007752) and an NIH/NINDS award (F31 NS108651). R.D. and T.A. are funded by NIH/NINDS R01 NS119615. T.A. is funded by NIH/NINDS grant no. 5K08NS096192. B.P. is funded by NIH/NINDS grant no. R01 NS034939. The UCSD Microscopy Core is funded by NINDS grant no. NS047101.
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C.E.D., T.A. and R.D. designed experiments and wrote the manuscript. C.E.D. performed and analyzed lineage tracing, single-cell and bulk RNA-seq, fibrosis reduction and IFN-γ experiments, and performed immunostaining and microscopy on EAE spinal cords. D.A. analyzed single-cell sequencing data. E.A.H. quantified the location of Col1a1-GFP+ cells in healthy and disease samples. R.N.S. analyzed fibrosis reduction experiments. K.K.H. performed and analyzed OPC in vitro experiments. L.P quantified the number of pericytes and immune cells in lesions over time. Y.C. and B.P. performed cuprizone experiments. T.A., C.O.L. and K.M.C. performed and analyzed bone marrow transplant and lineage tracing experiments. G.A.W. analyzed RNA-seq experiments, and S.P.J.F performed LPC surgeries.
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Extended data
Extended Data Fig. 1 Col1a1-GFP cell localization in health and following EAE.
a, Analysis of the number of Col1a1-GFP+ cells per total vascular length and smooth muscle actin (SMA) + vascular length in different CNS regions in healthy adult mice. CP = choroid plexus, SC = spinal cord white or grey matter, ± s.e.m., n = 4, Col1a1-GFP mice. b-d, Spinal cord sections from wild type mice in health or with EAE at 2, 5 or 10 days PSO were stained with Col1 (red), DAPI (blue) and CD11b (b, green), GFAP (c, green), or CD4 (d, green) Scale bars = 100 µm. e, Light sheet microscopy image of a Col1a1-GFP mouse in health (left) and one with EAE (right) 10 days PSO perfused with tomato lectin and optically cleared, scale bars = 200 µm. f, Confocal microscopy images of Col1a1-GFP spinal cords from health (left) and EAE (right) stained for CD31 in red, scale bars = 10 µm.
Extended Data Fig. 2 Fibrotic scarring is present in the LPC, but not cuprizone, model of demyelination.
a, Spinal cord sections from mice either 5, 7, or 14 days post LPC injection into the spinal cord were stained for either Col1 (green) and FluoroMyelin (red) or myelin basic protein (MBP) (green) and Cd11b (red). b, Brain sections in the area of the corpus callosum from GFAP/tTA;TRE/IFN-γ mice following cuprizone administration. GFAP/tTA;TRE/IFN-γ mice received doxycycline starting from birth, and half of the mice were taken off of doxycycline at 6 weeks of age (-dox) to induce the expression of IFN-γ in the CNS. Both the +dox and -dox groups were divided into two groups. The first group (3 wk) was given cuprizone for 3 weeks and then analyzed at the end of the 3 week period. The second group (8 wk) was given cuprizone for 5 weeks and then analyzed 3 weeks after completion of the cuprizone administration. All brains were stained for either Col1 (green) and FluoroMyelin (red) or myelin basic protein (MBP) (green) and Cd11b (red). Scale bar = 100 µM. c, Quantification of the proportion of the demyelinated area that is Col1+ from mice 7 days post LPC injection (n = 4) or mice 3 weeks post cuprizone administration with and without doxycycline (n = 3 each), ± s.e.m.
Extended Data Fig. 3 Col1a2CreERT and NG2CreERTM reporter expression.
Spinal cords of Col1a2CreERT;Rosa-tdTomato (a) or NG2CreERTM;Rosa-tdTomato (b) mice in health or EAE 10 d PSO were stained with CD31 in green and DAPI in blue. Scale bars = 100 µm. c, Spinal cords of Col1a2CreERT;Rosa-tdTomato;Col1a1-GFP mice in health or 10 days EAE PSO were imaged for both reporters. The percentage of GFP+ cells that were also positive for the tomato reporter in health and EAE is quantified in (d), ± s.e.m., n = 5 health, 4 EAE. e, Col1a2CreERT;Rosa-tdTomato mice were stained with NG2 in red, and the proportion of Col1a2CreERT+ cells that were NG2+ and NG2– were quantified in (f), ± s.e.m., n = 4. Scale bars = 100 µm.
Extended Data Fig. 4 Single-cell sequencing: cell purification and cluster expression.
a, Sample FACS plots of the purificaiton of Col1a1-GFP+ cells used for the single-cell sequencing analysis of Col1a1-GFP+ cells in health and EAE. b, UMAP plot of the single-cell RNA-seq dataset of Col1a1GFP+ cells in health and EAE with the individual sample identity labeled for each cell. c, Heat map depicting the expression of the 10 most differentially expressed genes in each cluster based on the logFC of the dataset. d, Heat map depicting the expression levels of genes specific to the labeled cell types, Olig = oligodendrocyte.
Extended Data Fig. 5 Effects of reducing fibrotic scar formation on immune cell infiltration, oligodendrocyte lineage cell populations and axon numbers.
a, Spinal cord sections from fHTK mice and controls were stained for DAPI (blue) and periostin (green), ER-TR7 (green), Col3 (green) and CD11b (red), or GFAP (green) and Cd11b (red). b, Spinal cord sections from fHTK mice and controls were stained for CD4 (red, left) and DAPI (blue) and the number of CD4+ cells per lesion area was compared between groups (right), p = 0.38 by Student’s two-tailed t-test, ± s.e.m., n = 21 control and 19 fHTK. c, Spinal cord sections from fHTK mice and controls were stained for CD8 (red, left) and DAPI (blue) and the number of CD8+ cells per lesion area was compared between groups (right), ± s.e.m., p = 0.22 by Student’s two-tailed t-test, n = 21 control and 19 fHTK. d, Spinal cord sections from fHTK mice and controls were stained for OLIG2 in green and CC1 in red, and the percent of OLIG2+ cells that were also CC1+ was quantified, ± s.e.m., p = 0.26 by Student’s two-tailed t-test e, Spinal cord sections from fHTK mice and controls were stained for neurofilament heavy polypeptide (NF) in green and CD11b in red, and the number of healthy, blebbed and total axons was quantified, ± s.e.m.. Scale bars = 100 µm.
Extended Data Fig. 6 Col1 reduces OPC migration, but not proliferation or differentiation, in vitro.
a, Representative images of OLIG2 (green) and PDGFRa (red) staining with EdU labeling (cyan) in rat OPC cultures on PLL, laminin, fibronectin, or collagen I after a 2 hour incubation in 10 uM EdU. b, Quantification of the percentage of EdU+ OPCs (OLIG2+PDGFRa+) for cultures represented in a., ± s.e.m., n = 9 replicates. c, Representative images of MBP (red) staining in rat OPC cultures on PLL, laminin, fibronectin, or collagen I three days after removal of PDGF-AA. Cell nuclei detected with DAPI (blue). d, Quantification of MBP+ cells over total cells (DAPI+) for cultures represented in c., ± s.e.m., n = 9 replicates. e, Representative images of PDGFRa (green) staining with DAPI in rat OPC cultures that had migrated through transwells coated with PLL, laminin, fibronectin, or collagen I following a 24 hour incubation. f, Quantification of PDGFRa+ cells on the underside of each transwell for the cultures represented in e., ± s.e.m., n = 9 replicates. Data displayed represent 3 replicates of 3 samples, each containing cells from three pooled postnatal day-7 rats. Comparisons were performed using one-way ANOVA with Sidak’s post hoc tests, ****p < 0.0001, ***p < 0.001, *p < 0.05. Scale bars = 100 µm.
Extended Data Fig. 7 RNA sequencing analysis of Col1a1-GFP+ cells from the spinal cord in health and EAE.
a-c, MA plots comparing the transcriptome of CNS fibroblasts in health with whole spinal cord tissue (a), CNS fibroblasts in health with CNS fibroblasts EAE D5 (b) or D10 (c) PSO with red dots signifying genes with FDR < 0.1. d, Pathway analysis using DAVID Bioinformatics Resources 6.8, NIAID/NIH, GOTERM_BP_DIRECT for genes with a log2 fold change greater than 2 for CNS fibroblasts EAE D5 PSO compared to CNS fibroblasts in health. e, CPM of collagen genes from the bulk sequencing of whole spinal cord tissue (Whole SC, n = 2), CNS fibroblasts from health (CNS Fibro Health, n = 3) CNS fibroblasts 5 days PSO (CNS Fibro EAE D5, n = 3) and 10 days PSO (CNS Fibro EAE D10, n = 3), ± s.e.m., *FDR < 0.05, **FDR < 0.01 to Whole SC, #FDR < 0.05, ##FDR < 0.01 to CNS Fibro Health. f, Heat map of the expression levels of the top genes differentially expressed in CNS Fibro Health (each expressed at least 1 CPM in each control sample) compared to the whole spinal cord by FDR.
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Dorrier, C.E., Aran, D., Haenelt, E.A. et al. CNS fibroblasts form a fibrotic scar in response to immune cell infiltration. Nat Neurosci 24, 234–244 (2021). https://doi.org/10.1038/s41593-020-00770-9
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DOI: https://doi.org/10.1038/s41593-020-00770-9
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