The role of platelets in the innate inflammatory response to TB.

Tuberculosis (TB) causes over 1.5 million deaths each year and there is increasing resistance to antibiotics used to treat the disease. There are few new anti-TB drugs, and new approaches to treating patients are required. In TB the immune system is overactive, and inflammation damages the lung tissues. This is a complex process involving a range of our own immune cells. Understanding this process better should help to identify opportunities to develop new treatments that reduce inflammation and tissue damage.

There is evidence that platelets, cells in the blood that are usually associated with blood clotting, are also important in regulating the immune system. They have been shown to be important in many different diseases that involve inflammation including cancer and arthritis, but very little is known about their role and their importance in TB.

Background work for this project shows that platelets increase levels of markers of inflammation that are produced by cells of the immune system when they are infected with TB. I also found that patients with TB have higher concentrations of platelet-derived factors in their blood compared to healthy controls, and these levels decreased when the patients were given TB treatment. The main idea behind my research, which is that platelets are important in regulating the human immune system's response to tuberculosis, is supported by these results.

My project has 2 parts:

1. I shall study the effect of platelets on production of markers of inflammation by monocytes, which are cells of the human immune system. Using techniques I have used in my previous work, I will obtain monocytes and platelets from blood taken from healthy donors. I will measure inflammatory markers produced by monocytes with and without platelets following TB infection, as well as measuring gene expression from these cells using a technique called qPCR. I will look at how platelets may affect the immune cells including studying the importance of direct contact between the platelets and the immune cells as a way of regulating inflammation and TB bacterial growth.

2. I shall then focus on the clinical importance of platelet activation in patients with TB before, during, and after anti-TB treatment. I will examine targets suggested by the work above to confirm that their effects can be demonstrated in patients as well as in cells in laboratory experiments. I shall recruit 120 adult TB patients (drug-sensitive disease only) and the same number of age- and sex-matched healthy controls. All subjects will be HIV-negative. I will collect clinical and X-ray data about the patients and any scarring left by infection with tuberculosis. Patients will have a test called spirometry to measure lung function. This will be done for TB patients at the end of their treatment, and in healthy controls for comparison. 30mls blood will be taken from patients before treatment, after 14 and 56 days, and at the end of treatment. The blood will be used to measure platelet activation and some will be frozen at -70C for later measurement of platelet factors, and the remainder used to obtain platelet and white blood cell counts. Some patients will have a procedure called a bronchoscopy as part of their clinical care. Fluid from the lungs will be obtained from these patients, and the cells and fluid tested for evidence of platelet activity. Together, these results will allow me to understand what is happening in terms of platelets and the immune response in TB patients.

In summary, this project will produce data about the role of platelets in TB. This is a new and exciting area that might affect TB treatment in the future. It will also provide me with an excellent clinical and laboratory science training that I need to progress as a clinical academic.

In tuberculosis (TB) innate immune inflammation leads to tissue destruction mediated by enzymes, particularly Matrix Metalloproteinases (MMPs). Better understanding of TB immunopathology will help identify novel host-directed therapies required in the era of increasing drug resistance. Platelets are immune regulators implicated in many inflammatory diseases, yet little is known about their role in TB. My preliminary data show that activated platelets increase monocyte MMP gene expression, secretion, and functional activity in TB. In patients, secretion of platelet factors (PF4, PDGF-BB, PTX3, and RANTES) are increased at baseline in cases compared to controls and decrease with treatment. These data support the hypothesis that platelets are key regulators of the innate inflammatory response and drive immunopathology in TB. My specific aims are to investigate:
1. Effects of platelets on monocyte differentiation and function in TB. I shall isolate cells from whole blood and measure secretion of inflammatory markers including cytokines and MMPs by Luminex assay and gene expression by qPCR. I will focus on the role of direct cell contact versus indirect signalling through mediators, particularly CD40L.
2. The clinical importance of platelet activation will be investigated in TB patients before, during, and after anti-mycobacterial treatment. Clinical data, sputum culture, and blood samples will be analysed from 120 TB-infected adult patients and 120 controls. Spirometry and chest X-ray will be performed to assess lung damage in TB patients versus controls. Whole blood will undergo FACS for evidence of platelet activity and plasma will be stored at -70C for analysis of CD40L and other inflammatory markers by Luminex. Samples will also be collected from patients undergoing bronchoscopy for analysis of platelet function, collagenase activity by FACS, and disease mechanisms. In summary, his project will define the role of platelets in innate inflammation in TB.

This study will greatly advance my development as an independent academic clinician. It will allow me to cultivate skills in a range of areas including laboratory techniques, data management and analysis, logistical skills necessary for project implementation, and personal skills such as management of a research team and allocation of resources. In addition, executing this project within a resource poor setting will have local benefits including strengthening existing research infrastructure and building capacity. I will be engaged in a bilateral process of learning and teaching which will benefit my colleagues as well as myself. In Peru I will be managing a full-time research nurse who will require training in data gathering and record-keeping, research practices and ethics, and specimen processing as well as trouble-shooting and logistical tasks. I will also be working closely with laboratory staff to whom I will provide support. I will also be able to impart acquired skills and knowledge to junior colleagues in the future.

The patients enrolled in the study will benefit from increased contact with clinical staff specialised in TB disease including myself, and from action to promote education and advocacy among patients, their families and communities, and healthcare staff involved in their care provision.

The scientific approach of the project will lead to an improved understanding of the immunopathology in TB disease, and the role of platelets in the immune system's complex signalling pathways. This knowledge is expected to feed into ongoing efforts to develop novel therapeutic approaches that would target the immune system and, together with anti-TB drugs, would enable improvements in morbidity and mortality associated with TB. The potential social and economic benefits of improved TB treatment would be far-reaching. The World Health Organisation has identified TB as a global public health priority and ongoing high rates of disease in both resource-poor and resource-rich settings, combined with the emergence of drug resistant TB, have driven a demand for new therapies. Development of new antibiotics with anti-TB effect has proven challenging: the lengthy incubation period means that drug development is slow and costs are high, and recent advances have unfortunately been limited by significant adverse effects. Novel therapeutic targets identified during this research may therefore be of interest to the pharmaceutical sector.

Finally, platelets are involved in the immunopathogenesis associated with a wide variety of systemic infectious and non-infectious diseases. Understanding their biology may therefore be of interest to researchers in a broad range of specialties including rheumatology, cardiology, and immunology.