Fig. 8: IAMs are a proliferating microglia subset.

a Pie chart showing proportion of cell-cycle genes expressed by IAMs that are highly expressed at specific stages of the cell cycle. Genes were assigned to specific stages of the cell cycle using annotations published in reference³⁵. See Supplemental Data 2 for a full list of genes included in the analysis and their cell cycle stage assignment. b Example flow cytometry plots of Ki67 staining within total microglia, and staining for IAMs markers CD206 and MSR1 in the Ki67^- and Ki67⁺ microglia populations. c Graph of frequency of total Ki67⁺ microglia at various time points post-infection (day 0, n = 7 mice; day 3, n = 3 mice; day 6, n = 6 mice; day 9, n = 5 mice). Data shown as mean +/- SEM and pooled from 2 independent experiments, with n = 5–8 mice analysed per time point. d Graph of frequency of Ki67⁺ positive cells within MSR1⁺CD206⁺ microglia (IAMs; filled bars) and MSR1^-CD206^- microglia (non-IAMs; open bars) at day 6 (n = 8 mice) and day 8 (n = 5 mice) post-infection. Data pooled from 2 independent experiments, with n = 8 (day 6 post-infection) or 5 (day 8 post-infection) mice analysed. Whiskers refer to the maximum/minimum values, the box refers to interquartile ranges, the centre line refers to the mean. Data analysed by two-way ANOVA, ****P < 0.0001, *P = 0.0103. e Example confocal images of Ki67 labelling of sections from uninfected or infected (day 8 post-infection) brains. An area of fungal infection is outlined with dashed white lines. White arrows denote Ki67⁺ proliferating cells. f Quantification of Ki67 staining relative to DAPI. Pixel density of each stain was calculated using ImageJ relative to section size. Each point represents a section analysed from individual mice (n = 2 mice analysed per group). g Brain and h lung fungal burdens from wild-type mice infected intranasally with C. neoformans at day 14 (n = 12 mice) and day 21 (n = 12 mice) post-infection. Data analysed by Mann Whitney U-test, **P = 0.0088. i Number of IAMs and (k) Ki67⁺ microglia in brains of wild-type mice infected intranasally with C. neoformans at day 14 (n = 12 mice) and day 21 (n = 12 mice) post-infection. j Frequency of Ki67⁺ cells within the IAMs and non-IAMs populations at day 14 post-infection (n = 12 mice). Data analysed by paired t-test, ****P < 0.0001. Each point represents an individual mouse, with lines denoting matched samples from the same animal. Intranasal data pooled from two independent experiments and analysed by unpaired t-tests. In all box-and-whisker plots, whiskers refer to the maximum/minimum values, the box refers to interquartile ranges, the centre line refers to the mean.