Fig 1: Interaction with BBB endothelial and ChP epithelial cells drives functional specification in MoCs. Relative mRNA expression of Nos2 and Arg1 genes in Munpolarized MoCs following 7h incubation with (A) BBB endothelial cells and (B) ChP epithelial cells. (A) Incubation with TNF-a+IFN-? BBB endothelial cells led to Nos2 upregulation in MoCs (Munstimul.Endothelium Mean = 1.74, SEM = 0.37; MIL-1ß Endothelium Mean = 2.74, SEM = 1.12; MTNF-a+IFN-? Endothelium Mean = 20.26, SEM = 10.6). In contrast, Arg1 relative mRNA expression was significantly increased in MoCs incubated with IL-1ß BBB endothelial cells (MIL-1ß Endothelium) as compared to Munpolarized cells (p < 0.0001), to Munstimulated Endothelium (p = 0.0001) and MTNF-a+IFN-? Endothelium (p = 0.0003). (B) MoCs incubated with IFN-? stimulated ChP epithelial cells (MIFN-? Epithelium) significantly upregulated Nos2 relative mRNA expression compared to Munpolarized (p = 0.013), to Munstimulated Epithelium (p = 0.014) and to MTNF-a Epithelium (p = 0.018). Similarly, MoCs incubated with unstimulated ChP epithelial cells (Munstimulated Epithelium) (p = 0.007) or with TNF-a ChP epithelial cells (MTNF-a Epithelium) (p = 0.01) displayed increased Arg1 mRNA expression. (C) mRNA expression of Csf2 and Csf3 in BBB endothelial cells. Following 16h of IL-1ß stimulation, BBB endothelial cells displayed a significantly increased Csf2 (one way ANOVA – F (2,6) = 8.97, p = 0.02) and Csf3 relative mRNA expression (one way ANOVA – F (2, 6) = 5.203, p = 0.05) compared to unstimulated and TNF-a+IFN-? BBB endothelial cells. The relative mRNA expression of Nos2 and Arg1 was calculated based on the 2-??Ct method using S16 (A, B) or Hprt (C) as reference genes. Data is presented as fold increase relative to Munpolarized condition (A, B) or to unstimulated condition (C); displayed are means and SEM from three individual experiments, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. A mean of the raw cycle threshold (Ct) values from the independent experiments performed are provided in Supplementary Table 3 and 4 . (D) mRNA expression of Arg1, Tpi1 and Gpi1 genes in MoCs following 7h incubation with BBB endothelial cells or ChP epithelial cells in the presence or absence of G-CSF and GM-CSF neutralizing antibodies. G-CSF + GM-CSF neutralizing antibodies (10µg/ml each) or isotype control (20µg/ml) were supplemented to MoCs incubated with BBB endothelial cells or ChP epithelial cells. A non-significant decrease in Arg1, Tpi1 and Gpi1 mRNA expression was observed in MoCs following blocking of G-CSF and GM-CSF in IL-1ß BBB endothelial cells (Arg1: IL-1ßIsotype Mean = 7.80, SEM = 3.29; IL-1ßG-CSF+GM-CSF Mean = 5.34, SEM = 1.45; Tpi1: IL-1ßIsotype Mean = 1.15, SEM = 0.02; IL-1ßG-CSF+GM-CSF Mean = 0.97, SEM = 0.12; Gpi1: IL-1ßIsotype Mean = 1.21, SEM = 0.11; IL-1ßG-CSF+GM-CSF Mean = 0.69, SEM = 0.23). Target genes were normalized to the reference gene S16 and the data is presented as fold increase relative to the unstimulated condition (2-??Ct). Displayed are means ± SEM from technical triplicates, 4 independent experiments. Raw cycle threshold (Ct) values from the independent experiments performed are provided in Supplementary Table 5 . (E) Arg1 relative mRNA expression in MoCs incubated for 7h with recombinant mouse G-CSF or GM-CSF proteins at different concentrations (10ng/ml, 50ng/ml or 100ng/ml). An increase in Arg1 expression was observed upon MoC incubation with GM-CSF (MoCs + 10ng Mean = 7.48, SEM = 4.36; MoCs + 50ng Mean = 9.0, SEM = 4.95; MoCs + 100ng Mean = 7.42, SEM = 3.71). Target genes were normalized to the reference gene Hprt and data presented as fold increase relative to the unstimulated condition (2-??Ct). Displayed are means ± SEM from technical triplicates, 3 independent experiments. Raw cycle threshold (Ct) values from the independent experiments performed are provided in Supplementary Table 6 .