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An evolutionary recent IFN-IL-6-CEBP axis is linked to monocyte expansion and tuberculosis severity in humans

Monocyte counts are increased during human tuberculosis (TB) but it has not been determined whether Mycobacterium tuberculosis (Mtb) directly regulates myeloid commitment. We demonstrated that exposure to Mtb directs primary human CD34+ cells to differentiate into monocytes/macrophages. In vitro myeloid conversion did not require type I or type II IFN signaling. In contrast, Mtb enhanced IL-6 responses by CD34+ cell cultures and IL-6R neutralization inhibited myeloid differentiation and decreased mycobacterial growth in vitro. Integrated systems biology analysis of transcriptomic, proteomic and genomic data of large data sets of healthy controls and TB patients established the existence of a myeloid IL-6/IL6R/CEBP gene module associated with disease severity. Furthermore, genetic and functional analysis revealed the IL6/IL6R/CEBP gene module has undergone recent evolutionary selection, including Neanderthal introgression and human pathogen adaptation, connected to systemic monocyte counts. These results suggest Mtb co-opts an evolutionary recent IFN-IL6-CEBP feed-forward loop, increasing myeloid differentiation linked to severe TB in humans.


Hematopoiesis, the development of different blood cell lineages from hematopoietic stem cells (HSCs), is a fundamental physiological process in vertebrates.
HSCs give rise to lineage-restricted progenitors that gradually differentiate into mature cells. Following cellular differentiation, single-lineage elements including erythrocytes, megakaryocytes, lymphocytes as well as myeloid cells such as monocytes and granulocytes circulate throughout the body performing diverse functions. While HSC development towards cellular lineages during homeostasis has been extensively studied (Hoggatt, Kfoury, & Scadden, 2016), the mechanisms by which how progenitors give rise to mature cells during stress responses are less comprehended. For instance, certain pathogens regulate production of blood cells by the bone marrow and it has been shown that fine-tuned regulation of cytokine-induced signals is required for differentiation of HSC into mature cell types (Kleppe et al., 2017;Mirantes, Passegue, & Pietras, 2014; C. C. Zhang & Lodish, 2008). For example, the protozoan parasite that causes kalazar, Leishmania donovani, inhabits the bone marrow of humans (Kumar & Nylen, 2012), targets bone marrow stromal macrophages (Cotterell, Engwerda, & Kaye, 2000) and induces differentiation of myeloid cells at the expense of lymphoid progenitors (Abidin, Hammami, Stager, & Heinonen, 2017;Cotterell et al., 2000). In the same line of evidence, after experimental exposure to Gram-negative bacteria, mice display increased amounts of bone marrow-derived neutrophils, through a G-CSF-C/EBPα dependent mechanism (Boettcher et al., 2014). Moreover, infection by intracellular bacteria has been shown to modulate production of circulating leukocytes involving IFNg-mediated pathways (Baldridge, King, Boles, Weksberg, & Goodell, 2010;MacNamara et al., 2011;Murray, Young, & Daley, 1998). Altogether, these studies indicate vertebrate hosts respond to infection by 'remodeling' cell lineage production, which are highly dependent upon the interplay of cytokine-induced hematopoiesis triggered during infection. Interestingly, recent reports have demonstrated hematopoietic stem/progenitor cells (HSPCs) may be infected by different classes of infectious agents such as viruses and bacteria, albeit at low efficiency (Carter et al., 2011;Kolb-Maurer, Wilhelm, Weissinger, Brocker, & Goebel, 2002). Therefore, since many pathogens may reach the bone marrow and provide microbial-HSC interactions, it is possible that, in addition to cytokines, pathogen recognition by progenitor cells directly regulate cell lineage commitment providing an anti-microbial defense system. In contrast, the Red Queen hypothesis (Van Valen, 1973) predicts such pathogens would benefit from cell lineage commitment to establish themselves into the host.
The human pathogen Mycobacterium tuberculosis (Mtb) has been recently detected in circulating HSCs (Lin -CD34 + ) from latent TB individuals (Tornack et al., 2017). Since Mtb can also gain access to the bone marrow during extra-pulmonary (Mert et al., 2001) as well as active pulmonary TB (Das et al., 2013), it has been suggested that the human bone marrow is a niche/reservoir for this bacterium during natural pathogen infection. However, whether interactions between Mtb and human CD34 + cells drive cellular differentiation has not been formally demonstrated. Interestingly, earlier (Rogers, 1928;Schmitt, Meuret, & Stix, 1977) and recent (Berry et al., 2010;Zak et al., 2016) studies have reported major changes in the peripheral myeloid cells such as increased blood counts and dysregulated "interferon transcriptional signature" during active TB. More specifically, several "interferon-stimulated genes" (ISGs) are modulated in circulating mature neutrophils and monocytes in active TB patients, which calls forth a possible role of such genes in TB pathogenesis (Berry et al., 2010;Dos Santos et al., 2018;Zak et al., 2016). In contrast, lymphocyte compartments were recently demonstrated to be contracted during progression from latent to active TB in humans (Scriba et al., 2017). Therefore, the observed changes in blood leukocytes could be a consequence of the interactions between Mtb and the bone marrow cellular environment. Thus, we hypothesized that Mtb regulates cellular differentiation of human HSPCs. By employing in vitro functional assays and integrated systems biology analysis of published available cohorts of healthy controls and TB patients, our study suggest that Mtb co-opts an evolutionarily recent IFN/IL-6/CEBP axis linking monocyte differentiation and disease severity. When compared to CD14 + cells, which are highly phagocytic cells, the MFI measurements within the CD34 + cell population were found to be ~4x lower ( Figure 1d).
These data suggest that PBMC CD34 + cells may be permissive to Mtb infection in vitro.
Similarly, purified cord blood derived CD34 + cells display comparable % and MFI as those seen in PBMC CD34 + cells (Figure 1-figure supplement 1b). Confocal microscopy analysis confirmed the presence of sparse intracellular mycobacteria in purified cord blood derived CD34 + cells at 4h post-infection (pi) (Figure 1-figure supplement 1c).
These findings raise the possibility that although human primary Lin -CD34 + cells can be infected by Mtb in vitro and in vivo (Tornack et al., 2017), this cell population may display intrinsic resistance to Mtb infection as it has been reported for other bacterial species (Kolb-Maurer et al., 2002). Next, we employed a purified cell culture system to investigate whether H37RV Mtb replicates in CD34 + cells in different time points. When sorted purified cord-blood CD34 + cells were exposed to Mtb H37Rv (MOI3) and cultivated in StemSpan TM SFEM II (Bodine, Crosier, & Clark, 1991;Keller, Ortiz, & Ruscetti, 1995), bacilli numbers exhibited a ~1.5-log growth at 5 days post infection (dpi) ( Figure 1e). As a control, purified CD14 + cells displayed higher bacterial proliferation than purified CD34 + cells over time (Figure 1-figure 1 supplement 1d). Together, these findings demonstrate that Mtb infects and replicates in primary human CD34 + cell cultures in vitro. While at 1 dpi bacilli were more associated with the surface of round cells (Figure 1f), at 5 dpi intracellular bacteria were associated with cells with abundant cytoplasm (Figure 1f), suggesting that Mtb-exposed cultures displayed increased frequencies of cells exhibiting morphological alterations over time ( Figure 1f, right panel).
Indeed, Giemsa staining (Figure 1g, arrows) presented higher frequency of cytoplasm richer cells in bacteria-exposed vs uninfected cell cultures (Figure 1g, right panel), thus suggesting that Mtb infection enhances cellular differentiation by CD34 + cells in vitro.

Live Mtb induces CD34 + cells towards myeloid differentiation and monocyte
output. Next, to investigate whether Mtb triggered cellular differentiation by human CD34 + cells, we evaluated differential expression of 180 transcription factors (TFs) associated with differentiation of distinct hematopoietic cells (Novershtern et al., 2011) in RNA-seq samples of Mtb-exposed purified cells ( source data 1). Next, we applied a network biology-built computational platform (Cahan et al., 2014) which, based on classification scores, can assess the extent to which a given population resembles mature cell types. Figure 2c (Figure 2-source data 1) shows that mRNA samples from Mtb-exposed CD34 + cells presented enrichment of monocyte/macrophage profiles, but not other mature cell populations such as lymphocytes or dendritic cells. Additionally, at 5 dpi, CD34 + cell cultures displayed increased frequencies of cells positive for CD11b (Figure 2d), a surface molecule expressed during myeloid differentiation (Hickstein, Back, & Collins, 1989;Rosmarin et al., 1989). Together, these data suggest that Mtb drives human primary CD34 + cells towards myeloid differentiation. We next employed flow cytometry to measure cell surface molecules previously associated with myeloid differentiation of human CD34 + cells (Cimato, Furlage, Conway, & Wallace, 2016;Gorczyca et al., 2011;Kawamura et al., 2017;Manz, Miyamoto, Akashi, & Weissman, 2002;Olweus, Lund-Johansen, & Terstappen, 1995). More specifically, we gated on CD34 + events and quantified % CD64 + CD4 + cells. Corroborating our hypothesis, Mtb enhanced the frequency of CD64 + CD4 + CD34 + cells (Figure 2e-h) at 5 dpi, but not CD10 + CD34 + (lymphoid) or  (Cimato et al., 2016;De Bruyn et al., 1995;Terstappen, Huang, Safford, Lansdorp, & Loken, 1991). When compared to their base line levels, we also observed an increased % CD4 + CD64 + CD34 + cells in Mtb-exposed bulk bone marrow samples from 2 healthy individuals (Figure 2figure supplement 1c, p = 0.08). In addition, frequencies of CD4 + CD64 + CD34 + cells were higher in cultures infected to live H37Rv Mtb than those exposed to heat-killed (HK) bacteria (Figure 2f,g). These results suggest that the observed cellular phenotype was mostly due to the activities of live pathogen and only partially to mycobacterial PAMPs such as TLR2 (Ara-LAM) or TLR9 (Mtb gDNA) agonists (Bafica et al., 2005;Underhill, Ozinsky, Smith, & Aderem, 1999), which induced CD38 and HLA-DR, but not CD4 and CD64 expression in CD34 + cells (Figure 2-figure supplement 1d). Importantly, increased frequency of CD4 + CD64 + CD34 + cells was also observed when cell cultures were exposed to a clinical isolate of Mtb (Figure 2h), ruling out a possible genetic factor associated with the laboratory strain H37Rv (Brites & Gagneux, 2015). Furthermore, Next, to investigate whether Mtb enhanced HSPC differentiation into mature myeloid populations (Kawamura et al., 2017;Lee et al., 2015;Manz et al., 2002), purified CD34 + cells were exposed to Mtb and 10d later, surface molecules were  (Figure 2c). Interestingly, in this in vitro culture system, CD14 + cells started to emerge at low levels at day 3 in both uninfected and Mtb-exposed cell cultures, while enhancement in monocyte frequency was observed in Mtb-stimulated cells at later time points, i.e. at 7 and 10 dpi.  (Figure 3j), which were previously associated with severe pulmonary TB (Balboa et al., 2011). Moreover, albeit not statistically significant, Mtb enhanced the frequency of CD16 + CD66b + neutrophils in the majority but not all samples tested (Figure 3b,f). In contrast, HK Mtb did not stimulate monocyte or neutrophil output associated markers were unchanged after exposure to live or HK Mtb. Altogether, these results suggest Mtb selectively favors the generation of monocytes and, to a lesser extent, neutrophils, by human CD34 + cells in vitro.

In vitro
Mtb-enhanced myeloid differentiation is mediated by IL-6R, but not type I or type II IFN signaling. Cytokines are important triggers of Lin -CD34 + differentiation in vivo and in vitro (Endele, Etzrodt, & Schroeder, 2014;Hoggatt et al., 2016;C. C. Zhang & Lodish, 2008) and Reactome pathway analysis of genes differentially expressed between Mtb-infected versus uninfected conditions displayed enrichment of "cytokine signaling in immune system" (  (Jansen et al., 1992;Zhao et al., 2014) which was confirmed in our system by the addition of exogenous IL-6 to CD34 + cells ( Figure 4-figure supplement 1a). Moreover, cytokine receptors, including IL6R, as well as their cytokine partners, containing IL6, were enriched in Mtb-exposed produce IL-6 following microbial stimuli (Allakhverdi & Delespesse, 2012), live Mtb also induced intracellular IL-6 production in Lin -CD34 + cells from bacteria-exposed PBMC 1d cultures ( Figure 4c). This was confirmed in 1d culture supernatants of purified CD34 + cells exposed to live Mtb which presented augmented levels of IL-6, but not IFN-g, IL-1b or TNF (

An IL6/IL6R/CEBPB gene module is enriched in the active TB transcriptome and
proteome. To investigate whether IL-6R signaling correlates with monocyte expansion and TB-associated pathology in vivo, we performed a comprehensive systems biology analysis integrating several large transcriptomic and proteomic data sets from published cohorts of healthy controls and patients with latent, active and disseminated TB (Berry et al., 2010;Hecker et al., 2013;Naranbhai et al., 2015;Novikov et al., 2011;Scriba et al., 2017) (Supplementary File 2). First, we used Ingenuity Pathway analysis (IPA) to determine IL-6/IL-6R upstream regulators in transcriptomes from publicly available CD14 + monocytes of active TB patients (Berry et al., 2010). . As shown in Figure 5a (top panel), IL6, IL6ST, IL6R and STAT3 were significantly enriched in transcriptomes of active TB monocytes, when compared to cells from healthy controls. As reported previously (Berry et al., 2010;Mayer-Barber et al., 2011;Novikov et al., 2011), STAT1 and IL1B were also confirmed as upstream regulators in active TB monocytes ( Figure   5a, top panel). We next examined potential genes share between IL6/IL6R and type I IFN signaling pathways in active TB monocytes. Strikingly, the two top upstream regulators in TB monocytes, IRF1 and STAT1 (Figure 5a, top panel), were the only genes in common between the TB monocyte gene signature (Berry et al., 2010), the "IL6/STAT3 pathway" and the "in vivo IFN-β" signature ( Figure 5a, Venn diagram, bottom panel), suggesting these genes might be regulated by both IL-6 and type I IFN during active TB in vivo. Since type I IFN and IL-6 share the ability to induce phosphorylation of both STAT1 and STAT3 (Ho & Ivashkiv, 2006), we ran gene set enrichment analysis (GSEA) (Subramanian et al., 2005) to identify potential overlapping downstream target genes in the whole blood "Berry TB" disease signature (Berry et al., 2010). In addition to the previously demonstrated type I IFN/STAT1 signature (Berry et al., 2010), the "IL6/STAT3" pathway was significantly enriched in this data set (FDR-corrected p<10 -4 , Supplementary File 2). Next, we defined protein signatures by overlapping the "Berry TB", the "IL6/STAT3" pathway with a published plasma proteome defining disease progression from latent to active TB (Scriba et al., 2017)  "CD34/myeloid", "IL6/STAT3" and "IFN/IL6-shared" pathways. Reanalysis of the published "Scriba Plasma TB" proteome set (Scriba et al., 2017) confirmed increased IL-6/STAT3 protein levels and changes in CD34/CD38 homeostasis, which were found to be early events in TB pathogenesis (Figure 5c, top panel). "IL6/STAT3" pathwayassociated proteins such as PLA2G2A, CRP, STAT3, IL-6 and CFB increased around 12 months before TB diagnosis (Figure 5c,d, top panels -orange circles and bars), which was concomitant with significant changes in the "IFN/IL6-shared" plasma markers CXCL10, IFNAR1 and MMP9 (Figure 5c,d top panels -blue circles and bars). The "IL6/STAT3" and "IFN/IL6-shared"pathways were also significantly enriched in a gene set recently linked to monocyte expansion in vivo, measured as monocyte:lymphocyte (ML) ratio (Naranbhai et al., 2015) (Supplementary File 2), and positively correlated with mycobacterial growth in vitro, thus connecting monocyte expansion and increased Mtb survival. Interestingly, the changes found in both the "IL6/STAT3" and "IFN/IL6-shared" pathways during development of TB disease preceded enrichment of the "ML ratio" gene set (Naranbhai et al., 2015) (6 months before diagnosis, p<0.05, Figure 5c, bottom panel and Figure 5d, top panel, gray bar) and reduction of the CD34/CD38 gene markers, in agreement with our in vitro model of Mtb-enhanced CD34 + differentiation (CD34 + → CD34 + CD38 + → CD14 + ). Moreover, fold changes were higher for "IL6/STAT3" pathway genes than for "IFN/IL6-shared" genes, and significantly higher than "ML ratio" genes (p<0.05) or "CD34/myeloid" differentiation genes (p<0.01) (Figure 5d bottom panel).
Together, these data suggest sequential activation of IL-6/IL-6R and IFN signaling pathways before monocyte expansion during TB disease progression in vivo, raising a possible link between these two events in disease pathogenesis. In support of this idea, CD34 + cells exposed to Mtb in vitro displayed increased levels of pSTAT1 as well as C/EBPβ (and a slight enhancement of C/EBPα), which are key TF regulators of ISGs and myeloid differentiation genes, respectively ( These results suggest that Mtb infection activates a gene module shared by both type I IFN and IL-6, linking downstream ISGs and CEBPs.

An IL6/IL6R/CEBP gene module correlates with monocyte expansion and TB
severity. TB pathogenesis is a convoluted process which interconnects mycobacterial dissemination, host inflammatory responses and systemic tissue pathology. To further investigate a potential link between this gene module (IL6/IL6R/CEBP) with disease severity and monocyte expansion in vivo, we first examined large transcriptomic data sets of "disseminated TB", which includes extrapulmonary and lymph node TB (GSE63548). The "IL6/STAT3" pathway was found to be significantly enriched among differentially expressed genes in both extrapulmonary (FDR p=10 -3 ) and lymph node TB 2), supporting its link with myeloid differentiation during homeostasis. Since the IL6/IL6R/CEBP gene module was correlated with both systemic disease dissemination and monocyte expansion, two processes associated with TB disease (Rogers, 1928;Schmitt et al., 1977), we next examined whether these genes might be connected to disease severity in a published cohort with detailed clinical parameters and transcriptome data (Berry et al., 2010). When compared to latent TB subjects, we observed that both the monocyte counts and ML ratio were significantly increased in active TB patients (Figure 5f). CEBPB transcripts positively correlated with ML ratio levels ( Figure 5g Santos et al., 2018). Taken together, these results indicate that the IL6/IL6R/CEBP gene module is a hub correlated with monocyte expansion during Mtb infection in vivo and is amplified in severe pulmonary and systemic disease.

Recent mammalian/primate genetic changes link an IFN/IL-6/IL-6R/CEBP axis to
monocyte expansion and TB pathogenesis in humans. The "type I IFN" signature found in active TB (Berry et al., 2010), shared with the IL-6/IL-6R-regulated gene set ( Figure 5), comprises a number of well-characterized ISGs with cross-species antiviral activity such as IRF1 and OAS. It has been reported that these ISGs have been undergoing strong purifying selection during primate evolution (Manry et al., 2011;Shaw et al., 2017), including recent Neanderthal introgression (Enard & Petrov, 2018;Quach et al., 2016). We thus undertook an evolutionary approach to investigate whether the IL6/IL6R/CEBP gene module and its partial overlap with type I IFN signaling is linked to monocyte expansion and TB severity. To do so, we performed a stepwise analysis, starting from early mammalian emergence (>100 million years ago (mya), over primate (>50 mya) and hominid evolution (>15 mya). We have also examined the recent human evolution including Neanderthal introgression (<100,000 years ago) and human pathogen adaptation (15,000-1,500 years ago), up to extant human genetic variation through analysis of large genome wide association studies (GWAS).
First, STRING network measurements of amino acid conservation and gene cooccurrence across mammalian and primate evolution revealed that IL-6, IL-6R and C/EBP family members C/EBPα, C/EBPβ and C/EBPδ differ substantially throughout primate evolution and even among closely related hominins (Pan troglodytes and Gorilla gorilla) (Figure 6a, heat map). In contrast, matched control molecules in the same STRING network (KLF5/NFKB1/MAPK1//STAT1/STAT3) remained largely conserved in most mammals, and even in birds and reptiles (Figure 6a, heat map and Figure 5-figure supplement 1d). Next, to investigate the biological consequence of the evolutionary differences in overlapping IL-6/IFN signaling, we reanalyzed cross-species type I IFN regulation from the 'mammalian interferome' database (Shaw et al., 2017). As expected, the conserved "IFN/IL6-shared" genes CXCL10/CXCL9/STAT1/STAT2 displayed higher fold-changes upon type I IFN treatment across all 10 species (from chicken to human, Figure 6b, top panel). Interestingly, the "IL-6/STAT3" pathway genes IL6, STAT3 and SOCS3 were also significantly upregulated while IL6R was significantly down-regulated Thus, transcriptional induction of CEBPB and CEBPD controlled by IL-6-and type I IFNsignaling appears as a relatively recent event in mammalian and primate evolution.

Genome-wide association studies (GWAS) connect the IL6/IL6R/CEBP
gene module with monocyte expansion in TB disease.
Since genetic susceptibility and transcriptional responses to intracellular pathogens have shown significant links to Neanderthal introgression in populations of European and Asian descent (Dannemann, Prufer, & Kelso, 2017;Quach et al., 2016), we next explored enrichment for introgression in the IL6/IL6R/CEBPB and "ML ratio" gene sets. As shown in Figure 6d (Figure 2-source data 1), eleven genes with Neanderthal introgression were significantly upregulated in our Mtb-exposed CD34 + cells transcriptome (enrichment p<0.0001). Of those, OAS1, OAS2 and MT2A transcripts had significantly higher effect sizes upon ML ratios, as compared to other introgressed genes (p<0.05) and to all other genes shown to regulate ML ratio in vivo (p<0.001, Figure 6d). This finding was confirmed in a recently published data set (n=198) of purified microbialexposed CD14 + monocytes from a Belgian cohort of European (EUB) and African (AFB) descendance, with documented presence or absence of Neanderthal introgression, respectively (Quach et al., 2016). Strikingly, 9 out of 11 introgressed genes enriched . Lastly, we examined whether myeloid differentiation genes identified in this study are found in GWAS TB susceptibility genes. A significant enrichment (p<0.0001) for differentially expressed genes from our Mtb-exposed CD34 + transcriptome and TB susceptibility GWAS/candidate genes (18 out of 172 genes, including IL6, STAT1 and CD14) was also observed ( Figure 6-figure supplement 1b).
Similarly, a significant (p<0.0001) overlap was found for the IL6/IL6R/CEPB module and TB genetic susceptibility (6 shared genes CD14, CXCL10, IL6, IL6R, IRF1 and STAT1, As ranked in Figure 7a, 24 out of 28 members of this gene module display a genome-wide, transcriptomic, proteomic or functional association to human TB, being strongest for IL6 and its downstream signaling TFs CEBPB and CEBPD, demonstrated in 5-6 independent data sets each. Collectively, our multi-level-based evidence suggests Mtb exploits an evolutionary recent IFN/IL-6/IL-6R/CEBP axis linked to monocyte expansion and human TB disease.

Discussion
Emerging evidence has suggested that Mtb establishes an infectious niche in the human bone marrow during active TB, which is associated with altered numbers of leukocytes in the periphery (Das et al., 2013;Mert et al., 2001;Naranbhai et al., 2015;Rogers, 1928;Schmitt et al., 1977;Tornack et al., 2017;Wang et al., 2015). In the present study, we observed that Mtb consistently stimulated myeloid differentiation molecules in CD34 + cell cultures from three different human tissues, namely: bone marrow, peripheral blood or cord blood samples. Employing a purified cord-blood derived CD34 + culture cell system, we observed that Mtb enhances IL-6R-mediated myeloid differentiation by human primary CD34 + cells in vitro. Importantly, IL-6/IL-6R downstream molecules such as C/EBPβ, C/EBPδ, STAT3 and their targets were significantly enriched in cell transcriptomes from active TB patients as well as were positively correlated with disease severity. Therefore, our data expands previous studies and raise a scenario in which Mtb skews myeloid development, mediated by IL-6/IL-6R signaling, as a key step in human TB pathogenesis.
While Mtb enhanced IL6 expression in purified CD34 + cell cultures from all donors, IFNA and IFNB mRNA were detected in some but not all donors. However, ISGs were highly enriched in the bacteria-exposed samples suggesting that although low/undetectable amounts of type I IFN were produced in infected cell cultures (Rodero et al., 2017), these cytokines were present in the cell culture ( Several independent studies have also indicated a detrimental role of "IFN and IFN-induced genes" during Mtb infection in human TB (Berry et al., 2010;Bustamante, Boisson-Dupuis, Abel, & Casanova, 2014;Dos Santos et al., 2018;Novikov et al., 2011;Scriba et al., 2017;G. Zhang et al., 2018) and murine models (Antonelli et al., 2010;Manca et al., 2001). Our results expand these previous studies, revealing a novel IL6/IL6R/CEBP gene module and its link to monocyte development, mycobacterial dissemination and TB disease severity. Furthermore, as evidenced by Scriba et al. (Scriba et al., 2017) and re-analyzed in the present study (Figure 5c,d), both IFN and IL6 pathways are early events in TB pathogenesis, detectable in the plasma proteome >6 months before diagnosis. Nevertheless, a pivotal role for IL6/IL6R signaling has not been evident from previous "omics" approaches. While IL-6 signaling partially overlaps with type I IFN responses (Figure 5a,b), possibly due to their shared ability to activate STAT1 and STAT3 (Ho & Ivashkiv, 2006), whole blood transcriptomic analyses in TB are predominated by an "IFN-inducible neutrophil signature" (Berry et al., 2010). Therefore, the high numbers of neutrophils in the blood possibly mask differential expression of other gene sets in less frequent populations, such as monocytes, monocyte subsets and, in particular, Lin -CD34 + cells.
Most ISGs present in the whole blood Berry TB signature (Berry et al., 2010), the Scriba TB plasma proteome (Scriba et al., 2017) and the Naranbhai et al. ML ratio gene set (Naranbhai et al., 2015) displayed cross-species type I IFN-induction throughout mammalian evolution (Figure 6b). Despite a shared STAT1/STAT3 activation by type I IFN and IL-6, homeostatic activation of C/EBPβ is mostly IL-6-specific, as evidenced by data mining and STRING analysis (Figure 6a), in keeping with its original description as NF-IL6 (Akira et al., 1990). Across species, CEBPB and CEBPD are among the highly variable ISG of the entire IFN/IL-6/CD34/myeloid gene set ( Figure 6b). Likewise, although type I IFN-induced IL6 and STAT3 transcription are conserved in all mammalian species studied, IFN-inducibility of CEBPB and CEBPD mRNA appeared to be recently acquired in primate evolution. In line with our observations, IL6, CEBPB and SPI1/PU1 genetic polymorphisms have been previously associated with TB susceptibility (G. Zhang et al., 2014;G. Zhang et al., 2012). In addition, a recently identified trans e-QTL (rs5743618) (Quach et al., 2016) in TLR1, a gene with peak Neanderthal introgression (Dannemann et al., 2017;Enard & Petrov, 2018;Hagai et al., 2018;Quach et al., 2016) has been associated to TB susceptibility in several populations worldwide (Barletta-Naveca et al., 2018;Naderi, Hashemi, Mirshekari, Bahari, & Taheri, 2016;Qi et al., 2015). Biological pathway analysis of genes significantly regulated in trans of rs5743618 revealed a significant enrichment of IL-6/STAT3 signaling and IL6 as the most connected gene (data not shown). In agreement, Mtb-induced macrophage IL-6 production, among other cytokines, can be predicted on the basis of strong genetic components as recently reported by (Bakker et al., 2018) in a large GWAS/immunophenotyping cohort study. By employing a data-driven multi-level analysis from large cohorts, we expand these previous observations and revealed significant genetic links shared between IL-6/IL-6R/CEBP signaling, CD34 + myeloid differentiation, monocyte homeostasis and TB susceptibility (compiled in Figure 7a).
Together, these findings favor the hypothesis that such genetic changes have undergone stepwise mammalian, primate and recent human selection, including Neanderthal introgression and worldwide population-specific pathogen adaptation.
In summary, our observations suggest that Mtb boosts myeloid differentiation by exploiting a feed-forward loop between IL-6 and type I IFN molecular networks, bridged by C/EBPβ (and C/EBPδ) (Figure 7b). Yet, further experiments will define the precise mechanisms of crosstalk between IFN, IL-6 and CEBP family members, specifically CEBPβ and CEBPδ, during natural Mtb infection. While this question merits direct investigation, nonetheless, the use of IL-6R blockade as an adjunct therapy to treat multi-drug resistant severe TB has been proposed (Okada et al., 2011;Zumla, Rao, Dodoo, & Maeurer, 2016). Thus, the present study provides evidence of a novel hostdirected target for therapeutic intervention in a major human disease.

Reagents. Mtb Ara-LAM was obtained from BEI Resources and used at 5 µg/mL. Mtb
H37Rv genomic DNA was obtained from 28 days colonies growing in Löwenstein-Jensen medium by CTAB method as previously described (Yamashiro et al., 2016).
Library based normalization was used to transform raw counts to RPKM and further normalized using the edgeR TMM normalization (3.10.0). Data were then transformed using the limma voom function (3.36.2), prior to batch correction using ComBat (sva 3.28.0). Negative binomial and linear model-based methods were used for differential expression analysis, using packages edgeR and limma packages. Differentially expressed genes (DEGs) were calculated with t-statistics, moderated F-statistic, and log-odds of differential expression by empirical Bayes moderation of the standard errors ( Supplementary File 4).
CellNet and CellRouter analysis. We applied CellNet to classify RNA-seq samples as previously described (Cahan et al., 2014). Raw RNA sequencing data files were used for (monocyte transcriptomes from active TB patients) or p<0.01 (differentially expressed genes in Mtb-exposed CD34 + cells and all publicly available datasets from GEO).
Differentially expressed genes were sorted into gene networks and canonical pathways, and significantly overrepresented pathways and upstream regulators were identified.

Fig. 2 | 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

Fig. 4 | Mtb enhances IL-6R-mediated myeloid differentiation in vitro.
Purified CD34 + cells were exposed to Mtb H37Rv (MOI3) for different time points and mRNA-seq was performed as described in the methodology section. a, Heatmap (z-score) of differentially expressed cytokine receptor genes. b, Heatmap (z-score) of differentially expressed cytokine genes. Shown is the average mRNA expression of three different donors from two independent experiments. c, PBMC from healthy donors were exposed to Mtb H37Rv, 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 Fig. 1a were analyzed for IL-6 MFI.

Additional Files
Supplementary File 1 -Reactome Pathways analysis of Mtb-exposed and control CD34+ cell transcriptomes.

Supplementary File 2 -Systems analysis (Ingenuity Pathway Analysis and Gene Set
Enrichment Analysis) of cohorts of healthy controls, patients with latent TB, active TB, disseminated TB, overlap with IL6/STAT3 signaling and myeloid development.
Supplementary File 3 -Human adaptation z-scores for IL6/IL6R/CEBP CD34 myeloid gene module and Gene set enrichment of Top500 human adaptation genes.
Supplementary File 4 -List of statistical methods used in the manuscript.