Fig. 1: In vivo CRISPR screen identifies cytokine receptors regulating macrophage polarization in neuroinflammatory lesions.
From: In vivo CRISPR screen reveals regulation of macrophage states in neuroinflammation
a, Scheme of the new experimental approach. Hematopoietic precursor (Hoxb8FL) cells were pre-differentiated in vitro for 48 h toward the myeloid lineage then i.v. injected into EAE-induced mice before disease onset. b, Histological characterization of the distribution of Hoxb8FL-derived and endogenous myeloid cells in EAE lesions in the spinal cord. Right, relative abundance of cells through lesions (depth from the spinal cord surface). N = 3 animals, 8, 11 and 11 lesions per animal. Scale bars, 10 μm. c, Representative flow cytometry plots of the transferred Hoxb8FL-derived cells (bottom row, green) compared to endogenous cells (top row, gray) in the spinal cord of the same mouse with EAE at the peak of disease. d, Quantification of the flow cytometry data. Top, myeloid cell markers; bottom, polarization markers. N = 4 animals for the myeloid marker panel and 3 animals for the polarization marker panel. e, Scheme of the pooled CRISPR screen targeting 109 cytokine receptors and downstream signaling genes with 15 control sgRNAs. f, Cytokine receptors regulating CNS migration, determined by comparing the spinal cord to the bone marrow compartment. g,h, Cytokine receptors regulating M-iNOS (g) or M-Arg1 (h) polarization, determined by comparing the single iNOS+ (g) or Arg1+ (h) spinal cord macrophages to the iNOS and Arg1 double-negative macrophages. Two-way analysis of variance (ANOVA) with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli for multiple comparisons, F = 1.98 × 10−16, P > 0.9999 used in b; multiple paired two-tailed t-tests or Wilcoxon tests with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli for multiple comparisons used in d; NS, P value > 0.05; *P value < 0.05, **P value < 0.01, ***P value < 0.001, **** P value < 0.0001; figures show the mean ± sd; not all genes included in the screen are shown for f–h. Asterisks indicate P values and false discovery rate (FDR) < 0.05 and absolute log2(fold change) > 3 times the s.d. of noise distribution (Methods).
