Fig. 4: Deletion of cytokine receptors differentially regulates lesion-promoting properties of macrophages in neuroinflammation.

a, Left, expression of each transcriptional signature related to lesion-promoting properties in Hoxb8FL control cells; right, phenotype of the KOs in Hoxb8FL-derived cells for the respective signature across macrophage clusters (Ma_Cx_1 to Ma_8) and overall (All) compared to the control cells. The shading/lines represent the distribution of log₂(fold change or FC) of the KO compared to the control for all the genes that make up the signature, with the thick line being the median (quantile 50) log₂(fold change), and the thinner overlaid shadows representing the lines of the adjacent quantiles: 40th and 60th quantiles of the log₂(fold change) delimit the darkest shadow flanking the median line, in steps of 10 until the 10th and 90th quantiles delimiting the lightest shadow at the edges. For statistical analysis, gene-set enrichment analysis (GSEA) was used (Methods). b, Scheme illustrating the detection of oxidized/reduced cellular states by excitation ratiometric imaging of the Grx1-roGFP2 sensor. c, Experimental design of the intravital imaging experiments based on the transfer of Hoxb8FL-derived myeloid cells expressing the Grx1-roGFP2 sensor. d, Representative images of the dorsal lumbar spinal cord along the midline vein, showing a neuroinflammatory lesion infiltrated by Hoxb8FL-derived myeloid cells expressing the Grx1-roGFP2 sensor. Filled white arrowheads indicate myeloid cells in an oxidized state, while outlined arrowheads indicate myeloid cells in a reduced state. Scale bars, 50 μm (overview image) and 10 μm (inset). e,f, Quantification of the redox state of Ifngr1-KO (e) or Tnfrsf1a-KO (f) Hoxb8FL-derived myeloid cells expressing the Grx1-roGFP2 sensor. Left, proportion of control (gray) or KO (purple) cells per animal that fall in each oxidation/reduction Grx1-roGFP2 ratio bin, where lower bin numbers mean low ratio; the whole range is divided into ten bins and each bin is equally sized; middle, cumulative distribution of the oxidized/reduced Grx1-roGFP2 ratios of all cells; right, percentage of cells per animal that show an oxidized/reduced ratio above the 75th percentile ratio of their experiment. N = 5 control and 4 Ifngr1-KO animals from two independent experiments; control, 883, 587, 95, 172 and 677, and Ifngr1-KO, 2,394, 1,585, 96 and 99 cells per animal analyzed (e); N = 4 control and 4 Tnfrsf1a-KO from two different independent experiments; control, 33, 128, 36 and 342, and Tnfrsf1a-KO, 145, 1612, 123 and 828 cells per animal analyzed (f). K–S, Kolmogorov–Smirnov statistic. In the box plots, the line shows median, the box extends from the first quartile (Q1) to the third quartile (Q3), and the whiskers extend to the smallest and largest values within 1.5 times the interquartile range from Q1 and Q3. In a, KO names marked by an asterisk at the top of the plots and black ‘All’ labels indicate that the phenotype of the signature for that KO versus control in the global Hoxb8FL-derived cells is significant. Blue indicates downregulated and red indicates upregulated in the KO compared to the control. Asterisks and black cluster name labels indicate the phenotype is significant for that cluster for the Hoxb8FL-derived KO versus the control. Significance was determined with the GSEA pathway analysis algorithm for a nominal (NOM) P value < 0.05 or an FDR q value < 0.25 and absolute normalized enrichment score (NES) > 1.5 (Methods); e,f, (left), Ordinary two-way ANOVA with a single pooled variance, F = 2.009, P = 0.0509 (e) and F = 0.2027, P = 0.9929 (f) for the interaction of genotype × bin. e,f (middle) K–S statistic. e,f, (right) one-tailed unpaired t-test. 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 ± s.d.

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