Fig. 2. Macrophage gene expression can be modeled as a function of activation, revealing a transitory stage of RELMɑ expressing cells.
(A) Macrophage activation-stage UMAP, showing an example of a fitted general additive model (GAM) for gene expression as a function of pseudotime.
(B) Top most significant GAM fits for genes associated with identified paths, showing single cells gene expression (dots - color matching activation-stage) as a function of pseudotime (x axis) for each activation path, with fitted models (black lines) and associated adjusted p values also shown.
(C) Fitted GAM models (colored lines matching activation paths) of gene expression of macrophage activation markers (y axis - fixed across all paths for each gene) as a function of pseudotime (x axis - specific to each path).
(D) Relative expression of RELMɑ coding gene Retnla and Ear2 shown as violin plots for each activation-stage (left) or as a fitted GAM models specifically for each activation path (right).
(E) Schematic view of experimental set up for adoptive bone marrow monocyte transfer from donor (CD45.1+) mice into the peritoneal cavity of naïve recipients (CD45.2+).
(F) Representative density flow cytometry plots (left) and quantification of percentage cells (right) expressing RELMɑ protein and mRNA (Retnla) in adoptively transferred bone marrow monocytes recovered from the peritoneal cavity of recipient mice at indicated times. Individual dots represent biological replicates from combined experiments (n = 10 across 2 repeats). Significant differences at each stage compared to day 2 are indicated (****: p value < 0.0001) based on single factor ANOVA analysis followed by Tukey Honest Significant Differences test.
(G) Quantification of percentage macrophages expressing RELMɑ 4 days post-adoptive cell transfer within host large peritoneal macrophages, adoptively transferred IL-4Rɑ sufficient (IL-4Rɑ+/+) or deficient (IL-4Rɑ−/−) monocytes (left), or in adoptively transferred large peritoneal macrophages (right). Individual dots represent biological replicates (n = 4–18). Significant differences at each stage compared to host macrophages are indicated (**: p value < 0.01; *: p value < 0.05; ns: p value > 0.05) or between transferred cells (ns: p value > 0.05) based on single factor ANOVA analysis followed by Tukey Honest Significant Differences test.
(H) Quantification of transferred RELMɑ sufficient (RELMɑ+/+) or deficient (RELMɑ−/−) macrophages 8 days post-adoptive cell transfer (top). Representative flow cytometry dot plots of indicated markers and quantification of proportion CD115+F4/80+ cells within adoptively transferred cells (bottom). Individual dots represent biological replicates (n = 5). Significant differences are indicated (***: p value < 0.001; *: p value < 0.05) based on Tukey Honest Significant Differences test.