Elsevier

Tuberculosis

Volume 89, Issue 6, November 2009, Pages 398-404
Tuberculosis

Immunological aspects
High granulocyte/lymphocyte ratio and paucity of NKT cells defines TB disease in a TB-endemic setting

https://doi.org/10.1016/j.tube.2009.07.004Get rights and content

Summary

Most people infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) actually maintain a strong immune response and are able to control bacterial growth (deemed latently infected (LTBI)), while approximately 10% progress to disease resulting in almost 2 million deaths per year. Determining the immune ‘footprint’ at specific stages of infection and disease will allow for better diagnostics, treatments and ultimately development of new vaccine candidates. In this study we performed multi-factorial flow cytometry on fresh blood from 56 TB cases, 46 Tuberculin Skin Test (TST) positive (LTBI) and 39 TST negative household contacts. We found a highly significant increase in granulocytes and decrease in B cells and invariant (Vα24 + Vβ11+) NKT cells in TB cases compared to TST+ contacts (p < 0.0001, p = 0.007 and p = 0.01 respectively) which were restored to LTBI levels following 6 months of TB treatment. Using support vector analysis, we found a combination of granulocyte and lymphocyte and/or NKT cell proportions allowed almost 90% correct classification into M. tuberculosis infection or disease. This work has important public health benefits in regards to diagnosis and treatment of TB in sub-Saharan Africa and in furthering our understanding of the requirements for protective immunity to TB.

Introduction

Tuberculosis (TB) is caused mainly by the intracellular pathogen, Mycobacterium tuberculosis (Mtb) and is one of the major health problems in developing countries.1 One unique feature about TB is that although one third of the world population is infected with Mtb, only about 5–10% of them progress to active disease, with higher rates among those co-infected with HIV.1 The majority remain healthy but latently infected (LTBI). Comparison of the immune system between individuals with active TB disease and those with LTBI will help to determine what is required for a protective immune response and will therefore enable specific immune responses to be targeted for future vaccine developments.2, 3 Determination of biomarkers for diagnosis of active TB disease, other than acid-fast bacilli (AFB) in sputum, is also essential in TB-endemic settings where less than 50% of individuals are positive2, 3 and in diagnostically difficult groups such as children, HIV co-infected subjects or those with extra-pulmonary TB where the current diagnostic tests on the market are unreliable.

A number of changes in the innate and adaptive arms of the immune system have been described with active TB disease. Neutrophils provide an innate immune resistance to TB most likely via chemokine secretion4 with risk of Mtb infection being inversely associated with neutrophil counts in contacts of pulmonary TB (pTB) patients.5 CD4+ T cells are specifically required for generating an optimal immune response to TB6, 7 mainly through production of IFN-γ which forms the basis of TB diagnostics to date.2, 8 Interestingly, with increasing disease severity, CD4 responses decrease7 and CD8+ T cells become more important although their contribution to human disease pathology is complicated and appears to reflect different stages of disease and bacterial burden.9, 10 A recent study demonstrated that a combination of moderate levels of both memory CD4+ T cells and CD8+ T cells were most effective in achieving protection; illuminating the requirement for vaccination strategies to target both subsets of cells.11 NKT cells provide a link between the innate and adaptive immune systems and have been shown to be decreased in TB disease12, 13 with higher levels seen in fast treatment responders.14 Other cell types such as γδ T cells,15 regulatory T cells,16 B cells17 and NK cells18 all appear to interact and control the eventual output response to TB.

In this study we performed multi-factorial analysis of the immune system using fresh, un-fractionated blood from subjects with active pTB disease (pre- and post-treatment) and compared this to Mtb-infected (TST+) and non-infected (TST−) household contacts. We found that increased granulocytes and decreased NKT cells could distinguish between TB disease and infection with almost 90% correct classification. These findings may have important public health implications in the development of new, simplistic diagnostic tools.

Section snippets

Participants

Fifty-six sputum smear-positive pTB cases and 85 of their Mycobacterium-exposed healthy household contacts were consecutively recruited as part of ongoing tuberculosis case contact studies at the MRC (UK) unit in The Gambia and the Gates Grand Challenge 6, Biomarkers for TB.3 Subjects were considered for inclusion if they were >18 years of age, were HIV-1 sero-negative. All cases and contacts underwent a clinical assessment, including a screen for malaria and inter-current illness. Of the

Study population

The median age of the TB cases was significantly higher than the contacts (28 compared to 23 years; p = 0.004). The majority (77%) of TB cases were male while 54% of contacts were female (p < 0.0001) and all were HIV negative. To allow for these possible confounding effects all analyses were adjusted for age and gender. Univariate results are presented in Table 1 with significant test results between TB cases and TST+ contacts shown based on a FDR of 10%. All subsets were significantly different

Discussion

This study provides a detailed analysis of the immune system in subjects with active TB compared to their latently infected household contacts with the aim of determining potential biomarkers associated with different stages of mycobacterial pathogenesis. Univariate analysis showed significantly elevated granulocytes and decreased B and NKT cell levels in TB cases compared to TST+ (likely Mtb-infected) household contacts. These parameters were all restored following 6-months of treatment.

Acknowledgements

We thank Mr. Ousman Secka and Mr. Gibril Bah for retrospective hematological details of adult pneumonia patients; field workers for sample collection, the TB Immunology laboratory staff, study participants and the Gambian National Tuberculosis and Leprosy Control programme for continuing collaboration. We would also like to thank Professor Sarah Rowland-Jones for critical review of the manuscript.

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    a

    Present address: Centre for International Health, University of Otago, New Zealand.

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