Validation of proteins associated with pathological damage in human tuberculosis granulomas: study protocol.

The presence of the Tuberculosis (TB) disease-causing pathogen, Mycobacterium Tuberculosis (Mtb), induces the development of a pathological feature termed granuloma, which the host uses to contain the bacteria. However, the granuloma may dissociate resulting in detrimental caseation of the lung. The disease contributes to a growing global burden of lung function challenges, warranting for more understanding of the TB-induced immunopathology. The current study aims to explore in detail host factors that drive pathological features of TB contributing to extensive lung tissue destruction. Lung tissue sections obtained from patients undergoing surgical resection will be processed and analyzed using histopathological assays including Immunohistochemistry, Immunofluorescence, Hematoxylin and Eosin staining and Laser Capture Microdissection. The findings will provide key host factors that associate with exacerbated lung immunopathology during TB.

Elevated IP-10 at the Protein and Gene Level Associates With Pulmonary TB.

There is an urgent need for accurate and sensitive diagnostic tools that can overcome the current challenge to distinguish individuals with latent tuberculosis infection (LTBI) from individuals with active tuberculosis (TB). Recent literature has suggested that a group of cytokines may serve as biomarkers of TB disease progression. Using a multiplex ELISA, we quantified 27 circulatory markers present within the unstimulated plasma of individuals in Durban, South Africa who were healthy (n=20), LTBI (n=13), or had active TB (n=30). RT-qPCR was performed to measure gene expression of the cytokines of interest, using RNA isolated from healthy (n=20), LTBI (n=20), or active TB (n=30). We found that at the protein level, IL-1RA, IL-6, and IP-10 were significantly more abundant in participants with active TB (p< 0.05) compared to those with LTBI individuals. IP-10 also showed the strongest association with active TB compared to healthy and LTBI at mRNA level. Our data shows that these proteins may serve as biomarkers of TB at both the protein and gene level.

Investigating neutrophil cell death in TB pathogenesis.

Background: Neutrophils are one of the major early role players in antimycobacterial immunity. Upon infection, neutrophils can undergo NETosis, a cell death characterized by release of neutrophil extracellular traps (NETs). The role of NETosis in TB progression remains poorly characterized. We aim to characterize mechanisms underlying NETosis during TB pathogenesis by identifying genes that drive the cell death, and to determine their potential as markers of disease progression in high-risk individuals. Finally, we intend to evaluate neutrophil associated genes as targets for host directed therapy to reduce pathological damage caused by NETosis. Methods: Quantitative PCR will be used to quantify expression of specific genes identified in the blood of individuals with active lung disease (n=30), compared to those from healthy (n=30) and latently infected individuals (LTBI) (n=30). In addition, temporal events associated with NETosis will be measured using live microscopy in a neutrophil in vitro model of Mycobacterium tuberculosis (Mtb) infection. Candidate genes found to be associated with NETosis will be targeted with pharmaceutical inhibitors. Conclusion: Genes associated with neutrophil mediated cell death may serve as potential biomarkers of pathological damage and disease progression, as well as targets for host-directed therapy.