Gene expression signatures identify biologically and clinically distinct tuberculosis endotypes.

BACKGROUND: In vitro, animal model and clinical evidence suggests that tuberculosis is not a monomorphic disease, and that host response to tuberculosis is protean with multiple distinct molecular pathways and pathologies (endotypes). We applied unbiased clustering to identify separate tuberculosis endotypes with classifiable gene expression patterns and clinical outcomes. METHODS: A cohort comprised of microarray gene expression data from microbiologically confirmed tuberculosis patients was used to identify putative endotypes. One microarray cohort with longitudinal clinical outcomes was reserved for validation, as were two RNA-sequencing (seq) cohorts. Finally, a separate cohort of tuberculosis patients with functional immune responses was evaluated to clarify stimulated from unstimulated immune responses. RESULTS: A discovery cohort, including 435 tuberculosis patients and 533 asymptomatic controls, identified two tuberculosis endotypes. Endotype A is characterised by increased expression of genes related to inflammation and immunity and decreased metabolism and proliferation; in contrast, endotype B has increased activity of metabolism and proliferation pathways. An independent RNA-seq validation cohort, including 118 tuberculosis patients and 179 controls, validated the discovery results. Gene expression signatures for treatment failure were elevated in endotype A in the discovery cohort, and a separate validation cohort confirmed that endotype A patients had slower time to culture conversion, and a reduced cure rate. These observations suggest that endotypes reflect functional immunity, supported by the observation that tuberculosis patients with a hyperinflammatory endotype have less responsive cytokine production upon stimulation. CONCLUSION: These findings provide evidence that metabolic and immune profiling could inform optimisation of endotype-specific host-directed therapies for tuberculosis.

DNA hypermethylation during tuberculosis dampens host immune responsiveness.

Mycobacterium tuberculosis (M. tuberculosis) has coevolved with humans for millennia and developed multiple mechanisms to evade host immunity. Restoring host immunity in order to improve outcomes and potentially shorten existing therapy will require identification of the full complement by which host immunity is inhibited. Perturbation of host DNA methylation is a mechanism induced by chronic infections such as HIV, HPV, lymphocytic choriomeningitis virus (LCMV), and schistosomiasis to evade host immunity. Here, we evaluated the DNA methylation status of patients with tuberculosis (TB) and their asymptomatic household contacts and found that the patients with TB have DNA hypermethylation of the IL-2/STAT5, TNF/NF-κB, and IFN-γ signaling pathways. We performed methylation-sensitive restriction enzyme-quantitative PCR (MSRE-qPCR) and observed that multiple genes of the IL-12/IFN-γ signaling pathway (IL12B, IL12RB2, TYK2, IFNGR1, JAK1, and JAK2) were hypermethylated in patients with TB. The DNA hypermethylation of these pathways was associated with decreased immune responsiveness with decreased mitogen-induced upregulation of IFN-γ, TNF, IL-6, CXCL9, CXCL10, and IL-1ß production. The DNA hypermethylation of the IL-12/IFN-γ pathway was associated with decreased IFN-γ-induced gene expression and decreased IL-12-inducible upregulation of IFN-γ. This study demonstrates that immune cells from patients with TB are characterized by DNA hypermethylation of genes critical to mycobacterial immunity resulting in decreased mycobacteria-specific and nonspecific immune responsiveness.

Evaluation of the QuantiFERON-Tuberculosis Gold Plus Assay in Children with Tuberculosis Disease or Following Household Exposure to Tuberculosis.

Interferon-gamma release assays are increasingly used in children to establish evidence of tuberculosis (TB) infection and to assist in the diagnosis of TB disease. The QuantiFERON-TB Gold In-Tube assay is being phased out in favor of a next-generation test, the QuantiFERON-TB Gold Plus (QFT-Plus) assay. The QFT-Plus assay is designed with two antigen tubes to differentially stimulate CD4+ and CD8+ T cells. The performance of this assay has been documented extensively in adults but has not yet been evaluated in children. Here, we compare the performance of the two assays in a cohort of 46 children exposed to TB and 12 children diagnosed with TB disease in Eswatini. The tests demonstrated excellent concordance in both TB disease (100% agreement, Cohen's kappa = 1) and TB infection (96% agreement, Cohen's kappa = 0.91). Most of the children with household exposure tested negative for TB infection by both tests, indicating the ongoing need for new tests for TB infection that can be easily implemented in TB high-burden settings at minimal cost.