Investigating neutrophil cell death in TB pathogenesis

Background

Tuberculosis is a disease caused by Mycobacterium tuberculosis (Mtb) and is responsible for approximately 1.5 million deaths annually¹. Understanding lung immunity during TB progression is important in identifying biomarkers. During initial TB infection, an immune structure known as a granuloma forms to control and prevent the spread of Mycobacterium tuberculosis². The granuloma is a highly organized immune structure composed of macrophages surrounded by a layer of epithelioid cells and multinucleated giant cells, with lymphocytic cuff³. Neutrophils are also found in the regions adjacent to caseum or necrotic regions of the granuloma⁴. Understanding the contribution of neutrophils to lung associated tissue damage is important in determining mechanisms that lead to TB associated pathology. Neutrophils are the most abundant cell subsets in the lung and are also amongst the first cells that are infected with Mtb⁴. Neutrophils play an important role in bacterial control during acute infection, however, they can release reactive oxygen species (ROS), deoxyribonucleic acids (DNA), myeloperoxidase, cathelicidins and S100 proteins that can result in excessive inflammation and damage to surrounding tissue^5,6. Netosis is the release of nuclear DNA and associated proteins i.e. histones, called neutrophil extracellular traps (NETS) from the cell in response to bacterial, viral or stress induced conditions⁷. The size of the NETS released is dependent on the size of the pathogen⁸, which is important in combatting infection. However, netosis has also been shown to contribute to tissue damage in various diseases^9–11. Studying neutrophils in a clinically relevant environment is challenging due to the short life-span and the various complex triggers of activation, including respiratory stress, damage-signalling or prior activation to pathogenic stimuli resulting in ROS associated activities¹².

Neutrophilia, which is a characterized by an abundance of neutrophils above what is considered to be a normal count, has been associated with TB and it is therefore important to understand mechanisms underlying neutrophil mediated contribution to disease pathogenesis^13,14. In previous findings, we reported that across cellular, caseous and cavitary granulomas, proteins associated with neutrophils were abundant in necrotic regions¹⁵ and in the border of necrotizing tissue⁵. However, whether neutrophils are directly causing caseation and how it is mediated remains a gap in our knowledge. Our project is therefore aimed at determining expression profiles of neutrophil associated genes in blood from healthy, LTBI and TB participants as potential markers of the disease. We also intend to investigate the role of neutrophils during TB pathogenesis by exploring, in-vitro, the dynamics of NETosis, in neutrophils infected with Mtb and to target drivers of the cell death as host directed therapies (HDT).

Materials and methods

Study size and ethics

A qualified nurse will enrol participants into the study after explaining the informed consent procedure according to good clinical health practices (Figure 1). Individuals who are newly diagnosed with active TB, determined as GeneXpert positive and who have not received treatment, will be recruited to the TB group of the study (n=30). Individuals who are healthy will be recruited to the healthy group as determined by their quantiferon (QFT) results. If the QFT result is positive those individuals will be recruited to the LTBI group (n=30) and those who are QFT negative will be recruited to the healthy group (n=30). Participants will be incentivised for participating in the study according to the ethics that have been approved through the University of KwaZulu-Natal. Participants will be informed that their biological samples will be stored for future use, including their genetic information, which will be used for research. A volume of twenty millilitres of blood will be used for neutrophil isolation. Plasma will be collected and stored at -80°C (Figure 2).

Figure 1. Schematic of the study design and experimental process that will be followed.

Figure 2. Schematic of plasma, neutrophil and RNA isolation from whole blood obtained from study participants.

NETosis quantification

A NETosis imaging kit (Cayman Chemical, Michigan, USA) will be used to quantify the amount of NETosis occurring in TB patients (n=4) compared to healthy participants (n=4). The assay will be performed as per manufacturer’s instructions. Briefly, 6 x 10⁶ neutrophils isolated on the same day, will be transferred to a clean tube and adjusted in 0.01 M PBS to 1 x 10⁶ cells/ml. A volume of six microlitres of Permeable Nuclear Red Reagent (5 mM) will be added to each well and incubated in the dark at 37°C for 15–30 minutes. A volume of 25 ml of PBS will be added to the cells, centrifuged at 250 x g and the supernatant aspirated. The cells will be resuspended in twelve microlitres (0.5 x 10⁶ cells/ml) of NETosis Imaging buffer (1x). A volume of twelve microlitres of Extracellular Nuclear Green Reagent (5 mM) will be added to the tube and mixed before the cells are seeded into the wells at 100 µl/well in a flat-bottom 96 well plate (Greiner Costar, Sigma-Aldrich, Germany). A volume of 100 µl of stimuli or NETosis Imaging Buffer (1x) will be added to each well for a final volume of 200 µl. The plate will be centrifuged at 250 x g to pellet the cells at the bottom of the plate. The BioTek cytation 5 (Biotek, Vermont, USA) which uses brightfield and fluorescence microscopy will be used to image activity every 30 min-1 hour for six to twelve hours at a temperature of 37°C and 5% CO₂. Green fluorescence indicates NETosis activity and red fluorescence is a marker for the nucleus (Figure 3). The neutrophils will be treated with optimized concentrations of various NETosis inhibitors depending on the chosen gene targets.

Figure 3. Images acquired with the Biotek Cytation 5 (1000 µM) to visualize and quantify NETosis.

Red indicates intact neutrophils. Green indicates neutrophils undergoing NETosis and extruding their cellular DNA. (Left) Negative control which is only healthy neutrophils with no stimulation. (Right) Positive control, PMA stimulated neutrophils.

Discussion

Proteomic analysis of lung tissue laser microdissections of various granulomas showed that proteins associated with neutrophils were abundant in necrotic regions of caseous and cavitary granulomas¹⁵. This indicated that neutrophils potentially contribute to necrotic damage observed in cavitary granulomas, due to their presence in these regions. This provides a potential link of the neutrophils with increased TB pathogenesis, hence the need to investigate their role during TB as potential target for therapy or as biomarkers. Identifying factors or mechanisms that contribute to lung tissue damage is vital in identifying markers of disease progression.

Neutrophils have been identified in the regions that border necrotizing tissue in other diseases¹⁷ and whether this is true for TB infection and granulomas remains to be determined. Recent studies have also identified the role of DNA-MPO^11,16 or histones complexes^10,18, ROS pathways⁹ and neutrophil elastase¹⁹ in tissue damage associated with various respiratory infections and as markers of disease progression. Studies in C3HeB/FeJ mice infected with Mtb have also shown that neutrophils contribute to necrosis and liquefaction of granulomas mediated by ROS events²⁰. These studies suggest that similar mechanisms mediated by neutrophil derived proteins and cell death activities may be driving TB lung pathology in humans.

NETosis proteins have been detected in the serum of individuals with acute respiratory distress syndrome associated pathology (as reviewed by 21). However, whether NETosis directly contributes to damaging pathology observed in TB granulomas is not well known. To validate this, our initial aim is to establish whether NETosis associated genes are present in the blood and are differentially expressed in LTBI, TB and healthy participants. In addition, we aim to determine whether expression of specific genes associated with NETosis correlates with the spectrum of disease observed during TB infection (as reviewed by 22,23). In addition, these unique gene expression profiles may be used to identify potential biomarkers of disease progression that may be used to diagnose TB at point of care.

Ongoing clinical trials are investigating the use of NETosis specific gene inhibitors to reduce tissue damage associated with NETotic events^24–28. Therefore, identifying potential targets for HDTs that may serve as adjunct therapies to current antibiotic regimens remains vital in combating the disease.

Due to the heavy TB disease burden in KZN, the study participants that will be enrolled in this study will provide important and relevant information regarding the tissue destruction mechanism that contributes to disease progression. A limitation of this study is that we will not be able to obtain matched blood samples from lung tissue donors. Another limitation of this study is that when performing the NETosis experiments it is difficult to distinguish between LTBI volunteers and those who are healthy in a timeous manner. This limits the ability of us to obtain a healthy blood volunteer to have an appropriate control.

The aim of this study is to identify potential biomarkers of TB disease progression that can lead to better diagnosis and ultimately, treatment of TB. We hope to characterize drivers of neutrophil mediated damage by developing a neutrophil in-vitro infection model. These findings may contribute to a better understanding of the neutrophil driven mechanisms that lead to damage often observed in the lungs of individuals with TB disease.

Data availability

Data will be stored and available through the Africa Health Research Institute according to the institutes protocols which insure participant privacy and rights protection.