Clinical and demographic data summary.

Demographic characteristics and magnitude of clinical induration of the skin at the site of TST or saline injection are summarised for all study subjects. Duration of anti-tuberculous drug treatment at the time of sampling and radiographic disease severity data are provided for individuals with pulmonary TB. *Peripheral blood samples were also taken from 50 of these individuals with pulmonary TB. Saline samples were obtained from 17 individuals with pulmonary TB and 32 healthy participants who were people with cured or latent TB, BCG vaccine recipients and healthy volunteers, described previously 10. Control blood samples from healthy individuals were described previously 13. **Applies only to participants with active TB receiving saline injections. CXR = chest x-ray.

The steady state human peripheral blood transcriptome reflects immune responses positively correlated with TB severity.

(A) Frequency distribution of the correlation coefficients for the relationship between the abundance of each of the 2620 transcripts which define the peripheral blood transcriptome derived from 50 individuals with pulmonary tuberculosis (TB) and radiographic disease severity. Coloured boxes highlight statistically significant correlations (p<0.05). (B) Reactome pathway enrichment among blood transcriptome genes whose expression is statistically significantly correlated with radiographic TB disease severity; corr = correlated. (C) Network diagram of statistically significant (FDR<0.05) upstream regulators (labelled nodes) coloured by their molecular function, of target gene modules whose expression is positively correlated with radiological TB severity. Size of the nodes for upstream regulators is proportional to -Log10 adjusted p value. Nodes were clustered using the Force Atlas 2 algorithm in Gephi (version 0.9.2).

Reduced type I interferon activity in the TST transcriptome is associated with increased severity of human pulmonary TB disease.

(A) Frequency distribution of the correlation coefficients for the relationship between the abundance of each of the 3222 transcripts within the TST transcriptome derived from 51 individuals with pulmonary TB and radiographic disease severity. Coloured boxes highlight statistically significant correlations (p<0.05). (B, C) Network diagrams of statistically significant (FDR<0.05) cytokines (B) and transcription factors (C) predicted to be upstream regulators (red) of target gene (blue) modules whose expression is negatively correlated with radiological TB severity. Size of the nodes for upstream regulators is proportional to -Log10 adjusted p value. Nodes were clustered using the Force Atlas 2 algorithm in Gephi (version 0.9.2). (D, E) The inverse relationship between average expression in each TST sample of two distinct, independently derived gene modules specific for type I interferon activity (Table S1) (D) with TB severity (E). r values and p values were derived from two-tailed Spearman rank correlations. CXR = Chest x-ray, MΦ = Macrophage, KC = Keratinocyte.

Increased severity of M. marinum infection in stat2 CRISPants.

(A) Schematic representation of the human canonical type I interferon signalling pathway (left) which is highly conserved in zebrafish (right). (B, C) The average expression of two largely non-overlapping (B) type I interferon inducible gene modules derived from independent data (Table S1) in genome-wide mRNA sequencing data from whole wild type embryos four days post-intravenous infection with 400 colony forming units (cfu) of M. marinum (Mm) compared with uninfected (UI) siblings (C). Data are from four independent experiments. (D) stat2 CRISPant (stat2-/-) and control ribonucleoprotein (RNP) injected (stat2+/+) zebrafish larvae four days post intravenous infection with 400 cfu M. marinum. Scale bar = 250 μm. Dashed white lines indicate the outline of each larva. (E) Survival of control and stat2 disrupted zebrafish larvae 1-4 days post infection with M. marinum. p value was derived from a log-rank (Mantel-Cox) test. (F-H) Comparison of integrated fluorescence (IF) (a surrogate for bacterial burden) (F), number of bacterial foci (G) and spatial distribution of bacteria (H), in stat2 CRISPants compared to control RNP injected siblings. Each data point represents an individual zebrafish larva, lines and error bars indicate the median and interquartile range. p values were derived from two-tailed Mann-Whitney tests. Data are representative of four experiments. dpi = days post-infection.

Reduced steady state neutrophil numbers in stat2 CRISPants.

(A) Representative images of three day post-fertilisation (dpf) control (upper panel) and stat2 CRISPant (lower panel) Tg(mpeg1:mCherry) zebrafish larvae delineated by the dashed white lines. Scale bar = 250 μm. (B) Integrated fluorescence (IF) as a surrogate for steady state macrophage numbers in three dpf stat2 CRISPants compared to siblings injected with scrambled ribonucleoproteins (RNPs). (C) Representative images of two dpf control (upper panel) and stat2 CRISPant (lower panel) Tg(mpx:eGFP) zebrafish larvae delineated by the dashed white lines. Scale bar = 250 μm. (D) Quantitation of baseline neutrophil numbers (represented by IF) in two dpf stat2 CRISPants and negative control RNP injected siblings. Each data point represents an individual zebrafish larva, lines and error bars indicate the median and interquartile range. p values were derived from two-tailed Mann-Whitney tests. Data are from three independent experiments.

Neutrophil recruitment to the site of sterile injury is not stat2 dependent.

(A) Representative images of eGFP-expressing neutrophils recruited to the site of injury 1, 6 and 24 hours following tailfin transection of three day post-fertilisation (dpf) stat2 CRISPant Tg(mpx:eGFP) zebrafish larvae and scrambled ribonucleoprotein (RNP) injected controls. Scale bar represents 100 μm. The white dashes indicate the outline of the tailfin. (B) Distance of neutrophils from the injury site at 1, 6 and 24 hours post-wound (hpw), derived by identifying the Sholl circle in which the maximum integrated fluorescence (IF), a surrogate for highest cell numbers, was detected in stat2 CRISPants and scrambled RNP injected siblings. (C-D) IF, a surrogate for total neutrophil numbers (C), and IF at the wound site, representing neutrophil numbers localized to the tailfin transection (D) are shown at 1, 6 and 24 hours following injury. On scatter plots data points represent individual zebrafish larvae and lines and error bars the median and interquartile range. On box and whisker plots horizontal lines represent median values, box limits indicate the interquartile range and whiskers extend between the 5th and 95th percentiles. p values were derived from two-tailed Mann-Whitney tests. * = p<0.05. Data are from three independent experiments. px = Pixels.

Reduced macrophage recruitment to the site of sterile injury in stat2 CRISPants.

(A) Representative images of mCherry-expressing macrophages recruited to the site of tailfin transection by 6 and 24 hours, in three day post-fertilisation (dpf) stat2 CRISPant Tg(mpeg1:mCherry) zebrafish embryos and controls. Scale bar represents 100 μm. The dashed white lines indicate the edge of the tailfin. (B) Comparison of the distance of macrophages from the site of injury at the indicated time points for stat2 CRISPants and control RNP injected siblings. (C) Integrated fluorescence (IF) a surrogate for macrophage numbers at the site of tailfin transection is shown at 1, 6 and 24 hours post-wound (hpw). On scatter plots data points represent individual zebrafish larvae and lines and error bars the median and interquartile range. On box and whisker plots horizontal lines represent median values, box limits indicate the interquartile range and whiskers extend between the 5th and 95th percentiles. p values were derived from two-tailed Mann-Whitney tests. * = p<0.05. Data are from three independent experiments. px = Pixels.

Reduced macrophage and neutrophil recruitment to M. marinum in stat2 CRISPants.

(A) Representative images of mCherry-expressing macrophages recruited to the hindbrain (dashed white outline) 18 hours post-injection (hpi) of PBS or 200 colony forming units (cfu) M. marinum in two day post-fertilisation (dpf) stat2 CRISPant Tg(mpeg1:mCherry) zebrafish larvae and scrambled ribonucleoprotein (RNP) injected controls. (B) Number of macrophages recruited to the hindbrain in response to PBS or M. marinum in stat2 CRISPants and negative control RNP injected siblings. (C) Representative images of eGFP-expressing neutrophils recruited to the hindbrain (dashed white outline) 6 hours after PBS or M. marinum injection (200 cfu) in two dpf stat2 CRISPant and control Tg(mpx:eGFP) zebrafish larvae. (D) Quantitation of neutrophils recruited to the site of M. marinum infection in stat2 CRISPants and control siblings. Scale bars represent 100 μm. Data points represent individual zebrafish larvae and lines and error bars the median and interquartile range. p values were derived from two-tailed Mann-Whitney tests. * = p<0.05. Data are from three independent experiments.