Figure 3. Analysis of macrophage pathways regulating the bimodal induction of the Type I IFN response.
(A) Induction of Cluster III is solely dependent on Trif signaling. iBMMs from WT, Tlr4−/−, Myd88−/− or Trif−/− mice were infected with S. typhimurium and expression of single cells was analyzed. Genes are arranged by a score summarizing their Myd88 or Trif dependence (MTR, left bars indicate distribution of scores for each cluster, Table S3A). (B) BMMs from Irf3−/− and Irf7−/− mice were infected with pHrodo-stained GFP-labeled S. typhimurium. Decreased induction of representative genes from Cluster III was evident in Irf3−/− cells, compared to increased induction levels in Irf7−/− cells. (Table S3B) (C) BMMs were infected with pHrodo-labeled GFP-labeled S. typhimurium, in the presence of BI2536 and BX795. While BI2536 inhibited mostly Cluster III genes but also genes from Cluster I and II, BX795 specifically inhibits only the induction of only Cluster III genes. (Table S3C) (D) Schematic representation of the gene regulatory networks that control the response of macrophages to S. typhimurium infection. The induction of the Type I IFN response is due to activation of Tbk1 and Irf3 in only a subset of infected cells. (E) Plots summarize the expression of each gene cluster in BMMs infected with live bacteria (top panel) or with LPS coated beads (bottom panel) using a weighted average of scaled expression values (x-axis) verses the frequency of single cells (y-axis). In contrast to the bimodal activation of the Type I IFN response in cells infected with live bacteria, there was a much higher proportion of cells that activated Cluster III among the cells that had taken up LPS coated beads.