Fig 2: PBMC cultures were exposed to Mtb H37Rv (MOI=3), HK Mtb or LPS (100 ng/mL) for 24h and intracellular IL-6 was detected by flow cytometry.Live CD34+Lin- events gated as in Figure 1A were analyzed for IL-6 MFI.

Fig 3: Mtb H37Rv infects human CD34+ cells and proliferates in cell cultures in vitro.PBMC from healthy donors were exposed to syto24-labeled Mtb H37Rv (MOI3, Figure 1—figure supplement 1a) for 4 hr. (a) Representative flow cytometry contour plots of gating strategy to analyze Mtb syto24 association in FVS-negative (live) CD34+ events and CD14+ events. (b) Live CD34+Lin- events gated in a were analyzed for Mtb-Syto24 MFI. Black line: Uninfected control. Blue, orange and purple lines represent samples from three different donors. (c) Frequencies and (d) MFI of Mtb syto24+ events in CD34+ or CD14+ events gates from uninfected or Mtb syto24-exposed bulk PBMCs. Results are means ± SEM of data pooled from three independent experiments, n = 10 healthy donors. ***p=0.001 between Mtb syto24 CD34+ vs CD14+ groups. (e,) Purified cord blood-derived CD34+ cells were exposed to Mtb H37Rv (MOI3) for different time points and CFUs from cell culture lysates were enumerated in 7H10 media. Results are means ± SEM of data pooled from five independent experiments, ***p=0.001 between 5d vs 4 hr groups. (f) Kinyoun staining of CD34+ cells after 1d and 5d of infection and quantification, as described in the methodology section, shown in the right panel. Arrows indicate cells associated with bacilli. Experiments shown are representative of two performed. **p=0.01 between 5d vs 1d groups. (g) Representative Giemsa staining of CD34+ cells of 5d-cultures and quantification, as described in the methodology section, shown in the right panel. Arrow indicates cytoplasm-rich cells in Mtb-infected cultures and uninfected cultures. Experiments shown are representative of two performed. *p=0.05 between Mtb vs uninfected groups.10.7554/eLife.47013.005Figure 1—source data 1.Raw data from Figure 1.

Fig 4: Evolutionary recent and human-specific genetic adaptation link IL6/IL6R/CEBP gene module with monocyte expansion and TB pathogenesis.(a) Heat map showing CEBPB network generated by STRING co-occurrence protein conservation scores across primates, mammals, birds and reptiles. Note only CEBPB and CEBPA differ strongly among hominids, while CEBPA/CEBPB/CEBPD vary significantly throughout primate and mammalian evolution, as compared to highly conserved STAT1/STAT3 (Figure 5—figure supplement 1d). (b) Top panel: Highly conserved type I IFN upregulation of ‘IFN/IL6-shared’ genes from humans to birds (derived from http://isg.data.cvr.ac.uk/) (Shaw et al., 2017), as compared to ‘IL6/STAT3’ and ‘CD34/myeloid differentiation’ genes. Bottom panel: CEBPB and CEBPD displays highest variation, and CXCL10 the lowest variation in type I IFN transcriptional regulation across human-mammalian-bird evolution. Inset, CEBPB and CEBPD selectively acquired type I IFN upregulation in humans (filled circles); ** p-value<0.01 and * p-value<0.05 represent CEBPB and CEBPD values, respectively for humans versus the other species. (c) ChipSeq analysis of STAT1-binding peaks in CEBPD (top panel), CEBPB (middle panel), CXCL9 and CXCL10 (bottom panel) in IFN-stimulated human monocytes, corresponding to regions with active chromatin (DNase Hypersensitivity Sites, DHS) and correlating with increased downstream transcription in CD14+ monocytes, as compared to purified CD34+ cells. Conservation analysis among >40 vertebrates (phyloP [Pollard et al., 2010], from chicken to human, analogous to Figure 5b) indicates STAT1 peaks are mostly conserved in CXCL9/CXCL10 (6/7) but not in CEBPD/CEBPB (3/11). (d) Top panel: overlap between human genes with significant Neanderthal introgression (Enard and Petrov, 2018; Quach et al., 2016), genes differentially expressed in Mtb-exposed CD34+ cells (CD34+ Mtb UP) and the ‘ML ratio’ gene set. Bottom left panel: OAS1, OAS2 and MT2A transcripts presented significantly higher effect sizes upon ML ratios, corresponding to monocyte expansion, as compared to other introgressed genes (p<0.05) and to all other genes shown to regulate ML ratio in vivo (p<0.001). Bottom right panel: normalized expression of introgressed genes found in CD34+Mtb UP (Venn diagram) in TLR1/2 agonist-treated monocytes from a cohort of matched Belgian individuals of European (EUB) vs. African (AFB) descendance, with documented presence or absence of Neanderthal introgression (Quach et al., 2016), respectively. p-value<0.05, ** p-value<0.01, *** p-value<0.001, **** p-value<0.0001.10.7554/eLife.47013.025Figure 6—source data 1.Raw data from Figure 6.

Fig 5: Live Mtb induces human CD34+ cells towards myeloid differentiation in vitro.Purified CD34+ cells from healthy donors (n = 3) were exposed to Mtb H37Rv (MOI3) for different time points and mRNA-seq was performed as described in methodology section. (a) Heatmap of the mRNA expression (z-score) of transcription factors involved in cell lineage commitment (Novershtern et al., 2011). (b) Signature score of data from a) by employing CellRouter analysis. (c) Heatmap from mRNA data of uninfected vs Mtb infected cultures analyzed by CellNet. (d) Purified CD34+ cells were exposed to Mtb H37Rv (MOI3) for 5 days and flow cytometry was performed. Graph represents frequencies of CD11b+ events in uninfected (open circles) vs Mtb-infected cultures (blue circles) from four independent experiments. **p=0.01 between Mtb and uninfected groups. (e) Purified CD34+ cells were exposed to Mtb H37Rv, Heat-killed (HK) Mtb H37Rv or Mtb clinical isolate 267 (Mtb-CS267) (MOI3) for 5 days and flow cytometry with the gating strategy was performed. (f) Representative contour plots show frequencies of CD4+CD64+ events in CD34+ events. CD34+CD4+CD64+ events of polled data from f) were plotted to generate bar graphs (g) and (h). Results are means ± SEM of data pooled from four independent experiments (g) and two independent experiments (h). (g) ** indicates p=0.01 between H37Rv vs uninfected or HK H37RV groups. (h) * indicates p=0.05 between Mtb-CS267 vs uninfected groups.10.7554/eLife.47013.009Figure 2—source data 1.Raw data from Figure 2.10.7554/eLife.47013.010Figure 2—source data 2.Counts matrix of RNAseq data of Mtb-exposed and control CD34+ cell transcriptomes.