Health & Medicine
June 8, 2022

Pulmonary tuberculosis: Biomarker discovery in the quest for new therapies

Pulmonary tuberculosis (TB) presents a significant health burden worldwide with treatments remaining suboptimal. In the pursuit of new or adjunctive therapies, Esmeralda Juárez and researchers at the Department of Research in Microbiology at the National Institute of Respiratory Diseases in Mexico City investigate the lungs’ immune-response mechanisms to TB infection. By sampling the breath of TB patients using a non-invasive technique, their research helps us understand disease mechanisms and identify host directed targets for therapeutic intervention through biomarker discovery.

Globally, tuberculosis (TB) from infection with Mycobacterium tuberculosis (MtB) causes more deaths than any other bacterial infectious disease. TB is highly prevalent in low- and middle-income countries and patients with active TB may present with fever, weight loss, and tiredness. Current treatments either directly target and kill MtB or they amplify immune-response mechanisms to fight the infection. However, treatments are not fully effective and new treatments as well adjunctive therapies (those that complement existing treatments) are needed.

Following infection, an inflammatory response ensues, and if this inflammation is not eventually resolved it can cause lung damage, pulmonary dysfunction, and/or pulmonary fibrosis (a disease marked by lung scarring and tissue damage). Consequently, TB can have profound effects on patients’ quality of life. Through their research, Dr Esmeralda Juárez and her team provide vital insights into lung immune-response mechanisms and work to identify potential therapeutic targets such as omega-3-derived metabolites which help reduce inflammation without affecting the body’s ability to destroy the bacteria. The research team also explore the utility of a non-invasive method to collect biomarkers and markers of inflammation and oxidative stress from the lungs in exhaled breath condensate (EBC).

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Their recent pilot study published in the journal Antioxidants revealed different quantities of biomarkers in exhaled breath of TB patients compared to healthy individuals. This suggests measurement of such biomarkers associated with lung damage can be used to both monitor patients over time to assess disease progression and to develop therapeutics.

The immune response and inflammation in TB
Pathogens need to be recognised by the body in order for the innate immune response to be triggered and the pathogen to be removed. Pattern recognition receptors such as Toll-Like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) receptors on cells such as macrophages (a type of white blood cell), have important roles in recognising foreign pathogens and launching an innate immune response to eliminate them. Macrophages have a key function of phagocytosis (the process of a cell ingesting and eliminating pathogens). Consequently, they evoke an immune response to help eliminate these pathogens. Macrophages are found throughout the body and are present in the lungs in the form of alveolar macrophages. These alveolar macrophages stimulate the release of pro-inflammatory cytokines which in turn recruit further immune cells, commencing the inflammatory process. Alveolar macrophages also have important anti-microbial activities and are vital in the defence and clearance of MtB from the lungs.

By regulating or stimulating the body’s immune response, the removal of Mtb from the lungs can be accelerated.

During infection, cytokines induce macrophages to release tumour necrosis factor alpha (TNF-α) which kills MtB by releasing reactive oxygen species (ROS) and reactive nitrogen species (RNS). An increase in ROS alters the redox state (the presence and balance of ROS) which causes oxidative stress. Eicosanoids (polyunsaturated fatty-acid-derived signalling molecules) have a critical role in balancing inflammation and resultant oxidative stress with the resolution of inflammation. The inflammatory response is needed to clear infection, but a prolonged inflammatory state and lack of antioxidant mechanisms can result in damage to lung tissue. Once the infection is eliminated, the inflammation must be reduced so that the cells and tissues can return to a homeostatic state. This requires pro-resolving eicosanoids which include the omega-3 fatty-acid-derived mediators such as Resolvin D1 and Maresin 1.

Figure showing the progression of pulmonary tuberculosis
Figure showing the progression of pulmonary tuberculosis. Alila Medical Media/Shutterstock.com

Adjunctive therapies for TB
Host directed therapy (modulating the host’s inflammatory responses to eliminate the bacteria as opposed to targeting it directly) is a key area of focus for Juárez’s research team. By regulating or stimulating the body’s immune response, the removal of MtB from the lungs can be accelerated. Innovative studies published by Juárez and colleagues in 2012 and 2014 elucidated the role of NOD2 and NOD1, respectively, in alveolar macrophage response to TB infection.

The 2012 study discovered a new function of NOD2 in human alveolar macrophages in inducing an antimicrobial response; specifically, stimulation of the antimicrobial peptide (LL37) activities. The team’s 2014 study provided novel data showing that NOD1 induces autophagy (when a cell recycles damaged components to ensure its own survival) and stimulates the release of proinflammatory cytokines in human alveolar macrophages in response to TB infection. Consequently, the study sparked an interest in the potential use of NOD1 ligands as a therapeutic target and use as immunomodulators (molecules which stimulate or modify the immune system) to treat TB.

Blood sample for biomarker test. Jarun Ontakrai/Shutterstock.com

The team went on to publish a study in 2016 showing the effectiveness of Loperamide (a drug usually used to treat diarrhoea) in inducing autophagy and destroying MtB in cells, suggesting its potential use as an adjunctive therapy for TB. Another finding by the research team is that pro-resolving mediators such as the lipids Resolvin D1 and Maresin 1 reduce the growth of MtB in cells and stimulate anti-inflammatory processes. These mediators, inducible by anti-inflammatory drugs such as aspirin, offer two main advantages in that they are not as strongly immunosuppressive as other anti-inflammatory treatments and that they can be obtained through diet. Such adjunctive therapies offer new therapeutic hope in the treatment of TB.

This pilot study has provided crucial information in the quest for biomarker discovery in TB and demonstrated the importance of measuring these from the lungs specifically.

Lung biomarker discovery in TB
Markers of a patient’s redox state or inflammation are usually measured in the serum or plasma of blood samples. However, in a recent pilot study by Juárez and colleagues, the team also searched for biomarkers from the lungs specifically as these are more representative of the local pulmonary response than blood markers. Bronchoscopy (a procedure to examine and sample the airways) is a more invasive method for sampling the lungs for biomarkers. Instead, exhaled breath condensate (EBC) was collected from both TB patients and healthy individuals breathing into an R-tube device (a device for collecting EBC) and the redox state and inflammation markers were then analysed.

Exhaled breath condensate was collected from both TB and healthy patients. SteLuk/Shutterstock.com

The results revealed reduced levels of the antioxidant Glutathione (GSH) in EBC, indicating alveolar oxidative stress in TB patients. In addition, 8-isoprostane (an eicosanoid marker of both oxidative stress and an insufficiency in antioxidants) was increased in the EBC of TB patients compared to healthy individuals. However, levels in serum were not different between health and disease. Neutrophils (a type of white blood cell) release nucleosomes to fight MtB infection as the infection is an indicator of oxidative stress. Raised nucelosome levels found in both EBC and serum of TB patients provides more evidence of oxidative stress in the lungs during TB infection. An increase in the pro-inflammatory eicosanoid Leukotriene B4 (LTB4) in the lungs of TB patients was also a novel finding of the study.

This pilot study provides crucial information in the quest for biomarker discovery in TB and demonstrates the importance of measuring these biomarkers from the lungs specifically. The successful measuring of such biomarkers gives researchers and clinicians an insight into disease and host response mechanisms and, if measured over time, could help us to better understand and determine disease progression. Furthermore, measurement of such biomarkers could be used both in the evaluation of treatment effects and potentially in the development of any new antioxidant-based therapies.

Vadi Fuoco/Shutterstock.com

Personal Response

Does your research team have plans to study the utility of EBC in patients with latent TB?

Yes, of course. We have several projects in mind. We want to understand the exhaled metabolites patterns in latent TB patients. Also, determine whether the exhaled metabolites patterns change along with the treatment in susceptible and multi-drug resistant TB patients. In either case, we could find signatures with the potential to aid in treatment success evaluations.

This feature article was created with the approval of the research team featured. This is a collaborative production, supported by those featured to aid free of charge, global distribution.

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