du Plessis, N. | bioRxiv Science

bioRxiv2022

Neutrophil degranulation, NETosis and platelet degranulation pathway genes are co-induced in whole blood up to six months before tuberculosis diagnosis

Mycobacterium tuberculosis (M.tb) causes tuberculosis (TB) and remains one of the leading causes of mortality due to an infectious pathogen. Host immune responses have been implicated in driving the progression from infection to severe lung disease. We analyzed longitudinal RNA sequencing (RNAseq) data from the whole blood of 74 TB progressors whose samples were grouped into four six-month intervals preceding diagnosis (the GC6-74 study). We additionally analyzed RNAseq data from an independent cohort of 90 TB patients with positron emission tomography-computed tomography (PET-CT) scan results which were used to categorize them into groups with high and low levels of lung damage (the Catalysis TB Biomarker study). These groups were compared to non-TB controls to obtain a complete whole blood transcriptional profile for individuals spanning from early stages of M.tb infection to TB diagnosis. The results revealed a steady increase in the number of genes that were differentially expressed in progressors at time points closer to diagnosis with 278 genes at 13-18 months, 742 at 7-12 months and 5,131 detected 1-6 months before diagnosis and 9,205 detected in TB patients. A total of 2,144 differentially expressed genes were detected when comparing TB patients with high and low levels of lung damage. There was a large overlap in the genes upregulated in progressors 1-6 months before diagnosis (86%) with those in TB patients. A comprehensive pathway analysis revealed a potent activation of neutrophil and platelet mediated defenses including neutrophil and platelet degranulation, and NET formation at both time points. These pathways were also enriched in TB patients with high levels of lung damage compared to those with low. These findings suggest that neutrophils and platelets play a critical role in TB pathogenesis, and provide details of the timing of specific effector mechanisms that may contribute to TB lung pathology. Author summaryMycobacterium tuberculosis (M.tb) causes tuberculosis (TB) and remains one of the leading causes of mortality due to an infectious pathogen. Human immune responses must be balanced to inhibit disease progression while limiting self-damage, however in defense against M.tb host responses have been implicated in lung damage and in driving progression of M.tb infection to severe lung disease. The identification of immune responses that are activated during the development of TB could provide potential targets for interventions that might suppress disease progression, and possibly limit tissue damage. Here we identify a subset of genes that function in several biological processes that are strongly activated in the late stages of TB development and in TB patients. The proteins encoded by these genes are known to degrade lung tissue and contribute to severe lung disease.

genomics↗

bioRxiv2021

Establishment of a Patient-Derived, Magnetic Levitation-Based, 3D Spheroid Granuloma Model for Human Tuberculosis.

Tuberculous granulomas that develop in response to Mycobacterium tuberculosis (M.tb) infection are highly dynamic entities shaped by the host immune response and disease kinetics. Within this microenvironment, immune cell recruitment, polarization and activation is driven not only by co-existing cell types and multi-cellular interactions, but also by M.tb-mediated changes involving metabolic heterogeneity, epigenetic reprogramming and rewiring of the transcriptional landscape of host cells. There is an increased appreciation of the in vivo complexity, versatility and heterogeneity of the cellular compartment that constitutes the tuberculosis (TB) granuloma, and the difficulty in translating findings from animal models to human disease. Here we describe a novel biomimetic in vitro 3-dimentional (3D) human lung granuloma model, resembling early "innate" and "adaptive" stages of the TB granuloma spectrum, and present results of histological architecture, host transcriptional characterization, mycobacteriological features, cytokine profiles and spatial distribution of key immune cells. A range of manipulations of immune cell populations in these granulomas will allow the study of host/pathogen pathways involved in the outcome of infection, as well as pharmacological interventions. IMPORTANCETuberculosis is a highly infectious disease, with granulomas as its hallmark. Granulomas play an important role in the control of M.tb infection and as such are crucial indicators for our understanding of host resistance to TB. Correlates of risk and protection to M.tb are still elusive, and the granuloma provides the perfect environment in which to study the immune response to infection and broaden our understanding thereof; however, human granulomas are difficult to obtain, and animal models are costly and do not always faithfully mimic human immunity. In fact, most TB research is conducted in vitro on immortalized or primary immune cells and cultured in 2D on flat, rigid plastic, which does not reflect in vivo characteristics. We have therefore conceived a 3D, human in vitro granuloma model which allows researchers to study features of granuloma-forming diseases, in an 3D structural environment resembling in vivo granuloma architecture and cellular orientation.

immunology↗