An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis

Abstract

Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, is a major cause of morbidity and mortality worldwide. Efforts to control it are hampered by difficulties with diagnosis, prevention and treatment. Most people infected with M. tuberculosis remain asymptomatic, termed latent TB, with a 10% lifetime risk of developing active TB disease. Current tests, however, cannot identify which individuals will develop disease. The immune response to M. tuberculosis is complex and incompletely characterized, hindering development of new diagnostics, therapies and vaccines. Here we identify a whole-blood 393 transcript signature for active TB in intermediate and high-burden settings, correlating with radiological extent of disease and reverting to that of healthy controls after treatment. A subset of patients with latent TB had signatures similar to those in patients with active TB. We also identify a specific 86-transcript signature that discriminates active TB from other inflammatory and infectious diseases. Modular and pathway analysis revealed that the TB signature was dominated by a neutrophil-driven interferon (IFN)-inducible gene profile, consisting of both IFN-gamma and type I IFN-alphabeta signalling. Comparison with transcriptional signatures in purified cells and flow cytometric analysis suggest that this TB signature reflects changes in cellular composition and altered gene expression. Although an IFN-inducible signature was also observed in whole blood of patients with systemic lupus erythematosus (SLE), their complete modular signature differed from TB, with increased abundance of plasma cell transcripts. Our studies demonstrate a hitherto underappreciated role of type I IFN-alphabeta signalling in the pathogenesis of TB, which has implications for vaccine and therapeutic development. Our study also provides a broad range of transcriptional biomarkers with potential as diagnostic and prognostic tools to combat the TB epidemic.

Figure 1. A distinct whole blood 393-gene transcriptional signature of active TB

393-transcripts differentially expressed in whole blood of active and latent TB patients and healthy controls a, Test Set. b, Validation Set (SA) profiles, ordered by hierarchical clustering (Spearman correlation with average linkage) creating a condition tree, upper horizontal edge of heatmap; study grouping (clinical phenotype) coloured blocks at each profile base. Heatmap rows = genes, columns = participants. c, Profiles were grouped according to radiographic extent of disease and the mean “Molecular Distance to Health” compared between groups (Methods) (Kruskal-Wallis ANOVA, Dunn's multiple comparison, *** = p <0.0001). d, Active TB patients at 0, 2 and 12 months after initiation of anti-mycobacterial treatment. The mean “Molecular Distance to Health” for each timepoint was compared (Friedman's repeated measures test, Dunn's multiple comparison). Horizontal bars indicate the median, 5^th and 95^th percentiles.

Figure 2. A distinct whole blood 86-gene transcriptional signature of active TB is distinct from other diseases

a, Comparison of 86-gene signature in patients with TB and other diseases normalized to their own controls; TB (Training, n=13; Control, n=12), TB (SA, n=20; Control = 12), Group A Streptococcus (Strep; n=23; Control=12), Staphylococcus (Staph; n=40; Control= 12), Still's disease (Still; n=31; Control=22), Adult (SLE; n=29; Control= 16) and paediatric SLE (pSLE; n=49; Control=11) patients. b, Expression levels of 86 gene signature after 2 and 12 months of treatment in TB patients.

Figure 3. Whole blood transcriptional signature of active TB reflects distinct changes in cellular composition and gene expression

a, Gene expression (disease versus healthy controls) of TB (Test Set) and different diseases mapped within a pre-defined modular framework. Spot intensity (red = increased, blue = decreased) indicates transcript abundance. Functional interpretations previously determined by unbiased literature profiling shown by colour-coded grid. Whole blood (Test Set active TB patients and controls) analysed by flow cytometry for (b) CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells and CD19⁺CD20⁺ B cells (c) CD14⁺ monocytes, CD14⁺CD16⁺ inflammatory monocytes and CD16⁺ neutrophils. Error bars = median, ** = p<0.01, * = p<0.05, Mann-Whitney test.

Figure 4. Interferon-inducible gene expression in active TB

Ingenuity Pathways analysis canonical pathway for interferon signalling symbol indicates gene function (legend on right). Transcripts over-represented in Test Set active TB patients shaded red a, Type II IFN-γ. b, Type I IFNαβ signalling. Transcript abundance of representative IFN-inducible genes in active TB (Test Set) from c, whole blood, d, separated blood leucocyte population. Transcript abundance/expression is normalised to the median of the healthy controls.