Katalin A. Wilkinson

An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis

Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, is a major cause of morbidity and mortality worldwide. Efforts to control it are hampered by difficulties with diagnosis, prevention and treatment. Most people infected with M. tuberculosis remain asymptomatic, termed latent TB, with a 10% lifetime risk of developing active TB disease. Current tests, however, cannot identify which individuals will develop disease. The immune response to M. tuberculosis is complex and incompletely characterized, hindering development of new diagnostics, therapies and vaccines. Here we identify a whole-blood 393 transcript signature for active TB in intermediate and high-burden settings, correlating with radiological extent of disease and reverting to that of healthy controls after treatment. A subset of patients with latent TB had signatures similar to those in patients with active TB. We also identify a specific 86-transcript signature that discriminates active TB from other inflammatory and infectious diseases. Modular and pathway analysis revealed that the TB signature was dominated by a neutrophil-driven interferon (IFN)-inducible gene profile, consisting of both IFN-γ and type I IFN-αβ signalling. Comparison with transcriptional signatures in purified cells and flow cytometric analysis suggest that this TB signature reflects changes in cellular composition and altered gene expression. Although an IFN-inducible signature was also observed in whole blood of patients with systemic lupus erythematosus (SLE), their complete modular signature differed from TB, with increased abundance of plasma cell transcripts. Our studies demonstrate a hitherto underappreciated role of type I IFN-αβ signalling in the pathogenesis of TB, which has implications for vaccine and therapeutic development. Our study also provides a broad range of transcriptional biomarkers with potential as diagnostic and prognostic tools to combat the TB epidemic.

Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells

BACKGROUND Identification of individuals latently infected with Mycobacterium tuberculosis is an important part of tuberculosis control. The current method, the tuberculin skin test (TST), has poor specificity because of the antigenic cross-reactivity of purified protein derivative (PPD) with M bovis BCG vaccine and environmental mycobacteria. ESAT-6 is a secreted antigen that is highly specific for M tuberculosis complex, but is absent from M bovis BCG. With an enzyme-linked immunospot (ELISPOT) assay for interferon gamma, we have identified ESAT-6-specific T cells as an accurate marker of M tuberculosis infection. METHODS We did a prospective, masked study of 50 healthy contacts, with varying but well defined degrees of exposure to M tuberculosis, who attended an urban contact-tracing clinic. We assessed and compared the efficacy of our assay and TST for detection of symptomless infected individuals by correlation of test results with the degree of exposure to an infectious index case. FINDINGS The ESAT-6 ELISPOT assay results had a strong positive relation with increasing intensity of exposure (odds ratio=9.0 per unit increase in level of exposure [95% CI 2.6--31.6], p=0.001), whereas TST results had a weaker relation with exposure (1.9 [1.0--3.5], p=0.05). By contrast, ELISPOT results were not correlated with BCG vaccination status (p=0.7), whereas TST results were significantly more likely to be positive in BCG-vaccinated contacts (12.1 [1.3--115.7], p=0.03). INTERPRETATION This new antigen-specific T cell-based assay could allow more accurate identification of symptom-free individuals recently exposed to M tuberculosis, and thereby help to improve tuberculosis control.

Enumeration of T Cells Specific for RD1-Encoded Antigens Suggests a High Prevalence of Latent Mycobacterium tuberculosis Infection in Healthy Urban Indians

Knowledge of the prevalence of latent Mycobacterium tuberculosis infection is crucial for effective tuberculosis control, but tuberculin skin test surveys have major limitations, including poor specificity because of the broad antigenic cross-reactivity of tuberculin. The M. tuberculosis RD1 genomic segment encodes proteins, such as early secretory antigenic target (ESAT)-6, that are absent from M. bovis bacille Calmette-Guérin (BCG) and most environmental mycobacteria. We recently identified circulating ESAT-6-specific T cells as an accurate marker of M. tuberculosis infection. Here, interferon-gamma-secreting T cells specific for peptides derived from ESAT-6 and a second RD1 gene product, CFP10, were enumerated in 100 prospectively recruited healthy adults in Bombay (Mumbai), India. Eighty percent responded to >/=1 antigen, and many donors had high frequencies of T cells that were specific for certain immunodominant peptides. In contrast, of 40 mostly BCG-vaccinated, United Kingdom-resident healthy adults, none responded to either antigen. This study suggests an 80% prevalence of latent M. tuberculosis infection in urban India.

Direct ex vivo analysis of antigen-specific IFN-gamma-secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals: associations with clinical disease state and effect of treatment.

The wide spectrum of clinical outcomes following infection with Mycobacterium tuberculosis is largely determined by the host immune response; therefore, we studied several clinically defined groups of individuals (n = 120) that differ in their ability to contain the bacillus. To quantitate M. tuberculosis-specific T cells directly ex vivo, we enumerated IFN-γ-secreting CD4 T cells specific for ESAT-6, a secreted Ag that is highly specific for M. tuberculosis, and a target of protective immune responses in animal models. We found that frequencies of circulating ESAT-6 peptide-specific IFN-γ-secreting CD4 T cells were higher in latently infected healthy contacts and subjects with minimal disease and low bacterial burdens than in patients with culture-positive active pulmonary tuberculosis (p = 0.009 and p = 0.002, respectively). Importantly, the frequency of these Ag-specific CD4 T cells fell progressively in all groups with treatment (p = 0.005), suggesting that the lower responses in patients with more extensive disease were not due to tuberculosis-induced immune suppression. This population of M. tuberculosis Ag-specific Th1-type CD4 T cells appears to correlate with clinical phenotype and declines during successful therapy; these features are consistent with a role for these T cells in the containment of M. tuberculosis in vivo. Such findings may assist in the design and evaluation of novel tuberculosis vaccine candidates.

Rapid detection of active and latent tuberculosis infection in HIV-positive individuals by enumeration of Mycobacterium tuberculosis-specific T cells.

Objectives: An accurate test for Mycobacterium tuberculosis infection is urgently needed. The tuberculin skin test (TST) lacks sensitivity, particularly in HIV-infected individuals, and has poor specificity because of antigenic cross-reactivity with Bacillus Calmette-Guérin (BCG) vaccination. ESAT-6 and CFP-10 are antigens expressed in M. tuberculosis, but not in Mycobacterium bovis BCG and most environmental mycobacteria. We investigated whether T cells specific for these antigens could serve as accurate markers of M. tuberculosis infection in an area of high tuberculosis and HIV prevalence. Methods: Using the rapid ex-vivo enzyme-linked immunospot (ELISPOT) assay for IFN-γ, we enumerated T cells specific for ESAT-6, CFP-10 and purified protein derivative (PPD) in blood samples from 50 Zambian tuberculosis patients, 75 healthy Zambian adults, and 40 healthy UK residents. TSTs were performed in 49 healthy Zambian adults. Results: All (100%; n = 11) and 90% (n = 39) of HIV-negative and HIV-positive tuberculosis patients, respectively, had detectable ESAT-6- or CFP-10-specific T cells. The ESAT-6/CFP-10-based ELISPOT assay was positive in 37 out of 54 HIV-negative healthy Zambians, suggesting a 69% prevalence of latent M. tuberculosis infection. Fewer HIV-positive Zambians possessed ESAT-6/CFP-10-specific T cells, but the impact of HIV infection was less on this assay than on the PPD-based ELISPOT or TST. Conclusion: The ESAT-6/CFP-10-based ELISPOT assay detects active tuberculosis in HIV-positive individuals with high sensitivity. It is more specific, and possibly more sensitive, than PPD-based methods of detecting latent M. tuberculosis infection, and may potentially improve the targeting of isoniazid preventative therapy to HIV-positive individuals with latent tuberculosis infection.

IFN-gamma- and TNF-independent vitamin D-inducible human suppression of mycobacteria: the role of cathelicidin LL-37.

Vitamin D deficiency is associated with susceptibility to tuberculosis, and its biologically active metabolite, 1α,25 dihydroxyvitamin D3 (1α,25(OH)2D3), has pleiotropic immune effects. The mechanisms by which 1α,25(OH)2D3 protects against tuberculosis are incompletely understood. 1α,25(OH)2D3 reduced the growth of mycobacteria in infected human PBMC cultures in a dose-dependent fashion. Coculture with agonists or antagonists of the membrane or nuclear vitamin D receptors indicated that these effects were primarily mediated by the nuclear vitamin D receptors. 1α,25(OH)2D3 reduced transcription and secretion of protective IFN-γ, IL-12p40, and TNF in infected PBMC and macrophages, indicating that 1α,25(OH)2D3 does not mediate protection via these cytokines. Although NOS2A was up-regulated by 1α,25(OH)2D3, inhibition of NO formation marginally affected the suppressive effect of 1α,25(OH)2D3 on bacillus Calmette Guérin in infected cells. By contrast, 1α,25(OH)2D3 strongly up-regulated the cathelicidin hCAP-18 gene, and some hCAP-18 polypeptide colocalized with CD14 in 1α,25(OH)2D3 stimulated PBMC, although no detectable LL-37 peptide was found in supernatants from similar 1α,25(OH)2D3-stimulated PBMC cultures. A total of 200 μg/ml of the active peptide LL-37, in turn, reduced the growth of Mycobacterium tuberculosis in culture by 75.7%. These findings suggest that vitamin D contributes to protection against TB by “nonclassical” mechanisms that include the induction of antimicrobial peptides.

Neutrophil-mediated innate immune resistance to mycobacteria

Neutrophils contain antimicrobial peptides with antituberculous activity, but their contribution to immune resistance to tuberculosis (TB) infection has not been previously investigated to our knowledge. We determined differential white cell counts in peripheral blood of 189 adults who had come into contact with patients diagnosed with active TB in London, United Kingdom, and evaluated them for evidence of TB infection and capacity to restrict mycobacterial growth in whole-blood assays. Risk of TB infection was inversely and independently associated with peripheral blood neutrophil count in contacts of patients diagnosed with pulmonary TB. The ability of whole blood to restrict growth of Mycobacterium bovis bacille Calmette Guérin and Mycobacterium tuberculosis was impaired 7.3- and 3.1-fold, respectively, by neutrophil depletion. In microbiological media, human neutrophil peptides (HNPs) 1-3 killed M. tuberculosis. The neutrophil peptides cathelicidin LL-37 and lipocalin 2 restricted growth of the organism, the latter in an iron-dependent manner. Black African participants had lower neutrophil counts and lower circulating concentrations of HNP1-3 and lipocalin 2 than south Asian and white participants. Neutrophils contribute substantially to innate resistance to TB infection, an activity associated with their antimicrobial peptides. Elucidation of the regulation of neutrophil antimicrobial peptides could facilitate prevention and treatment of TB.

Ex Vivo Characterization of Early Secretory Antigenic Target 6–Specific T Cells at Sites of Active Disease in Pleural Tuberculosis

Presence of early secretory antigenic target-6 (ESAT-6)-specific, interferon- gamma -secreting T cells in blood accurately marks tuberculosis infection. In tuberculous pleural effusions from 10 patients with tuberculosis, these cells were concentrated a mean of 15-fold (standard deviation, +/-6-fold), relative to their level in peripheral blood (P=.014), and displayed rapid effector function. Such cells were absent in 8 control patients with nontuberculous pleural disease. The recruitment of ESAT-6-specific T cells to inflamed tuberculous tissue demonstrates their function in vivo and suggests a novel way to diagnose tuberculous pleuritis.

Reciprocal seasonal variation in vitamin D status and tuberculosis notifications in Cape Town, South Africa

Vitamin D deficiency is associated with susceptibility to tuberculosis (TB) in HIV-uninfected people in Europe, but it is not known whether such an association exists among HIV-infected people in subtropical Africa. We conducted a cross-sectional study to determine whether vitamin D deficiency was associated with susceptibility to active TB in HIV-uninfected (n = 196) and HIV-infected (n = 174) black Africans in Cape Town, South Africa. We also investigated whether there was evidence of seasonal variation in vitamin D status and TB notifications in this setting over an 8-y period. Vitamin D deficiency (serum 25-hydroxyvitamin D [25(OH)D] <50 nmol/L) was present in 232 (62.7%) of 370 participants and was associated with active TB in both HIV-uninfected (odds ratio = 5.2, 95% confidence interval: 2.8–9.7; P < 0.001) and HIV-infected (odds ratio = 5.6, 95% confidence interval: 2.7–11.6; P < 0.001) people. Vitamin D status varied according to season: The mean serum 25(OH)D concentration was highest in January through March and lowest in July through September (56.8 vs. 30.7 nmol/L, respectively; P < 0.001). Reciprocal seasonal variation in TB notifications was observed: The mean number of TB notifications per quarter for Cape Town in 2003 to 2010 was lowest in April through June and highest in October through December (4,222 vs. 5,080; P < 0.001). Vitamin D deficiency is highly prevalent among black Africans in Cape Town and is associated with susceptibility to active TB both in the presence and absence of HIV infection. Reciprocal seasonal variation in serum 25(OH)D concentration and TB notifications suggests that seasonal variations in vitamin D status and TB incidence in this setting are causally related.

Transcriptional blood signatures distinguish pulmonary tuberculosis, pulmonary sarcoidosis, pneumonias and lung cancers.

Rationale New approaches to define factors underlying the immunopathogenesis of pulmonary diseases including sarcoidosis and tuberculosis are needed to develop new treatments and biomarkers. Comparing the blood transcriptional response of tuberculosis to other similar pulmonary diseases will advance knowledge of disease pathways and help distinguish diseases with similar clinical presentations. Objectives To determine the factors underlying the immunopathogenesis of the granulomatous diseases, sarcoidosis and tuberculosis, by comparing the blood transcriptional responses in these and other pulmonary diseases. Methods We compared whole blood genome-wide transcriptional profiles in pulmonary sarcoidosis, pulmonary tuberculosis, to community acquired pneumonia and primary lung cancer and healthy controls, before and after treatment, and in purified leucocyte populations. Measurements and Main Results An Interferon-inducible neutrophil-driven blood transcriptional signature was present in both sarcoidosis and tuberculosis, with a higher abundance and expression in tuberculosis. Heterogeneity of the sarcoidosis signature correlated significantly with disease activity. Transcriptional profiles in pneumonia and lung cancer revealed an over-abundance of inflammatory transcripts. After successful treatment the transcriptional activity in tuberculosis and pneumonia patients was significantly reduced. However the glucocorticoid-responsive sarcoidosis patients showed a significant increase in transcriptional activity. 144-blood transcripts were able to distinguish tuberculosis from other lung diseases and controls. Conclusions Tuberculosis and sarcoidosis revealed similar blood transcriptional profiles, dominated by interferon-inducible transcripts, while pneumonia and lung cancer showed distinct signatures, dominated by inflammatory genes. There were also significant differences between tuberculosis and sarcoidosis in the degree of their transcriptional activity, the heterogeneity of their profiles and their transcriptional response to treatment.