Jeroen Maertzdorf

Common patterns and disease-related signatures in tuberculosis and sarcoidosis

In light of the marked global health impact of tuberculosis (TB), strong focus has been on identifying biosignatures. Gene expression profiles in blood cells identified so far are indicative of a persistent activation of the immune system and chronic inflammatory pathology in active TB. Definition of a biosignature with unique specificity for TB demands that identified profiles can differentiate diseases with similar pathology, like sarcoidosis (SARC). Here, we present a detailed comparison between pulmonary TB and SARC, including whole-blood gene expression profiling, microRNA expression, and multiplex serum analytes. Our analysis reveals that previously disclosed gene expression signatures in TB show highly similar patterns in SARC, with a common up-regulation of proinflammatory pathways and IFN signaling and close similarity to TB-related signatures. microRNA expression also presented a highly similar pattern in both diseases, whereas cytokines in the serum of TB patients revealed a slightly elevated proinflammatory pattern compared with SARC and controls. Our results indicate several differences in expression between the two diseases, with increased metabolic activity and significantly higher antimicrobial defense responses in TB. However, matrix metallopeptidase 14 was identified as the most distinctive marker of SARC. Described communalities as well as unique signatures in blood profiles of two distinct inflammatory pulmonary diseases not only have considerable implications for the design of TB biosignatures and future diagnosis, but they also provide insights into biological processes underlying chronic inflammatory disease entities of different etiology.

Human gene expression profiles of susceptibility and resistance in tuberculosis

Tuberculosis (TB) still poses a profound burden on global health, owing to significant morbidity and mortality worldwide. Although a fully functional immune system is essential for the control of Mycobacterium tuberculosis infection, the underlying mechanisms and reasons for failure in part of the infected population remain enigmatic. Here, whole-blood microarray gene expression analyses were performed in TB patients and in latently as well as uninfected healthy controls to define biomarkers predictive of susceptibility and resistance. Fc gamma receptor 1B (FCGRIB)was identified as the most differentially expressed gene, and, in combination with four other markers, produced a high degree of accuracy in discriminating TB patients and latently infected donors. We determined differentially expressed genes unique for active disease and identified profiles that correlated with susceptibility and resistance to TB. Elevated expression of innate immune-related genes in active TB and higher expression of particular gene clusters involved in apoptosis and natural killer cell activity in latently infected donors are likely to be the major distinctive factors determining failure or success in controlling M. tuberculosis infection. The gene expression profiles defined in this study provide valuable clues for better understanding of progression from latent infection to active disease and pave the way for defining predictive correlates of protection in TB.

Biomarkers of Inflammation, Immunosuppression and Stress with Active Disease Are Revealed by Metabolomic Profiling of Tuberculosis Patients

Although tuberculosis (TB) causes more deaths than any other pathogen, most infected individuals harbor the pathogen without signs of disease. We explored the metabolome of >400 small molecules in serum of uninfected individuals, latently infected healthy individuals and patients with active TB. We identified changes in amino acid, lipid and nucleotide metabolism pathways, providing evidence for anti-inflammatory metabolomic changes in TB. Metabolic profiles indicate increased activity of indoleamine 2,3 dioxygenase 1 (IDO1), decreased phospholipase activity, increased abundance of adenosine metabolism products, as well as indicators of fibrotic lesions in active disease as compared to latent infection. Consistent with our predictions, we experimentally demonstrate TB-induced IDO1 activity. Furthermore, we demonstrate a link between metabolic profiles and cytokine signaling. Finally, we show that 20 metabolites are sufficient for robust discrimination of TB patients from healthy individuals. Our results provide specific insights into the biology of TB and pave the way for the rational development of metabolic biomarkers for TB.

Functional correlations of pathogenesis-driven gene expression signatures in tuberculosis.

Tuberculosis remains a major health threat and its control depends on improved measures of prevention, diagnosis and treatment. Biosignatures can play a significant role in the development of novel intervention measures against TB and blood transcriptional profiling is increasingly exploited for their rational design. Such profiles also reveal fundamental biological mechanisms associated with the pathology of the disease. We have compared whole blood gene expression in TB patients, as well as in healthy infected and uninfected individuals in a cohort in The Gambia, West Africa and validated previously identified signatures showing high similarities of expression profiles among different cohorts. In this study, we applied a unique combination of classical gene expression analysis with pathway and functional association analysis integrated with intra-individual expression correlations. These analyses were employed for identification of new disease-associated gene signatures, identifying a network of Fc gamma receptor 1 signaling with correlating transcriptional activity as hallmark of gene expression in TB. Remarkable similarities to characteristic signatures in the autoimmune disease systemic lupus erythematosus (SLE) were observed. Functional gene clusters of immunoregulatory interactions involving the JAK-STAT pathway; sensing of microbial patterns by Toll-like receptors and IFN-signaling provide detailed insights into the dysregulation of critical immune processes in TB, involving active expression of both pro-inflammatory and immunoregulatory systems. We conclude that transcriptomics (i) provides a robust system for identification and validation of biosignatures for TB and (ii) application of integrated analysis tools yields novel insights into functional networks underlying TB pathogenesis.

Enabling biomarkers for tuberculosis control.

Accelerated control of tuberculosis (TB) requires better control measures. Biomarkers, which reliably diagnose active TB or even predict risk of disease progression in individuals, could facilitate rapid diagnosis and treatment of TB patients and allow preventive measures for latently infected individuals with a high risk of TB. Moreover, biomarkers could speed up clinical trials with novel drug and vaccine candidates. Three platforms of global biomarker profiling will be described, with an emphasis on the most recent achievements: transcriptomics, proteomics and metabolomics. Moreover, we will discuss the need for computational analyses to make the best use of the plethora of data generated by biomarker research. Aside from their potential prognostic and diagnostic value, biomarkers could provide deeper insight into pathological processes underlying disease, and hence form the basis for novel intervention measures that target host molecules and pathways. We propose that biosignatures, which discriminate active TB from both latent infection and uninfected status, as well as from other diseases, will become available within the next decade. However, simple, low-cost biomarker-based point-of-care diagnosis will probably not be achieved in the next few years.Accelerated control of tuberculosis (TB) requires better control measures. Biomarkers, which reliably diagnose active TB or even predict risk of disease progression in individuals, could facilitate rapid diagnosis and treatment of TB patients and allow preventive measures for latently infected individuals with a high risk of TB. Moreover, biomarkers could speed up clinical trials with novel drug and vaccine candidates. Three platforms of global biomarker profiling will be described, with an emphasis on the most recent achievements: transcriptomics, proteomics and metabolomics. Moreover, we will discuss the need for computational analyses to make the best use of the plethora of data generated by biomarker research. Aside from their potential prognostic and diagnostic value, biomarkers could provide deeper insight into pathological processes underlying disease, and hence form the basis for novel intervention measures that target host molecules and pathways. We propose that biosignatures, which discriminate active TB from both latent infection and uninfected status, as well as from other diseases, will become available within the next decade. However, simple, low-cost biomarker-based point-of-care diagnosis will probably not be achieved in the next few years.

Differential gene expression of activating Fcγ receptor classifies active tuberculosis regardless of human immunodeficiency virus status or ethnicity

New diagnostics and vaccines for tuberculosis (TB) are urgently needed, but require an understanding of the requirements for protection from/susceptibility to TB. Previous studies have used unbiased approaches to determine gene signatures in single-site populations. The present study utilized a targeted approach, reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), to validate these genes in a multisite study. We analysed ex vivo whole blood RNA from a total of 523 participants across four sub-Saharan countries (Ethiopia, Malawi, South Africa, and The Gambia) with differences in TB and human immunodeficiency virus (HIV) status. We found a number of genes that were expressed at significantly lower levels in participants with active disease than in those with latent TB infection (LTBI), with restoration following successful TB treatment. The most consistent classifier of active disease was FCGR1A (high-affinity IgG Fc receptor 1 (CD64)), which was the only marker expressed at significantly higher levels in participants with active TB than in those with LTBI before treatment regardless of HIV status or genetic background. This is the first study to identify a biomarker for TB that is not affected by HIV status or geo-genetic differences. These data provide valuable clues for understanding TB pathogenesis, and also provide a proof-of-concept for the use of RT-MLPA in rapid and inexpensive validation of unbiased gene expression findings.

Concise gene signature for point‐of‐care classification of tuberculosis

There is an urgent need for new tools to combat the ongoing tuberculosis (TB) pandemic. Gene expression profiles based on blood signatures have proved useful in identifying genes that enable classification of TB patients, but have thus far been complex. Using real‐time PCR analysis, we evaluated the expression profiles from a large panel of genes in TB patients and healthy individuals in an Indian cohort. Classification models were built and validated for their capacity to discriminate samples from TB patients and controls within this cohort and on external independent gene expression datasets. A combination of only four genes distinguished TB patients from healthy individuals in both cross‐validations and on separate validation datasets with very high accuracy. An external validation on two distinct cohorts using a real‐time PCR setting confirmed the predictive power of this 4‐gene tool reaching sensitivity scores of 88% with a specificity of around 75%. Moreover, this gene signature demonstrated good classification power in HIV+ populations and also between TB and several other pulmonary diseases. Here we present proof of concept that our 4‐gene signature and the top classifier genes from our models provide excellent candidates for the development of molecular point‐of‐care TB diagnosis in endemic areas.

The dual role of biomarkers for understanding basic principles and devising novel intervention strategies in tuberculosis

There is great need for better control measures for tuberculosis (TB). High‐throughput analyses, such as transcriptomic and metabolic profiling, offer a promising path toward clinically useful biosignatures. With the help of biomarkers, it will be possible not only to reliably perform diagnosis but also to gain a better understanding of the disease process and, in the future, even predict the onset of disease in infected individuals. Biomarkers based on transcriptomic and metabolic profiles as well as on cytokine composition provide important insights into the basic biological principles of TB and give an opportunity to reliably distinguish TB patients from healthy individuals. Use of biomarkers for point‐of‐care diagnosis, however, is still a distant goal, which to achieve will require extensive analysis of TB biosignatures across different cohorts and a combination of different platforms.

Platelets Direct Monocyte Differentiation Into Epithelioid-Like Multinucleated Giant Foam Cells With Suppressive Capacity Upon Mycobacterial Stimulation

Background. Epithelioid, foam, and multinucleated giant cells (MNGCs) are characteristics of tuberculosis granulomas, yet the precise genesis and functions of these transformed macrophages are unclear. We evaluated the role of platelets as drivers of macrophage transformation in mycobacterial infection. Methods. We employed flow cytometry and microscopy to assess cellular phenotype and phagocytosis. Immune assays allowed quantification of cytokines and chemokines, whereas gene microarray technology was applied to estimate global transcriptome alterations. Immunohistochemical investigations of tuberculosis granulomas substantiated our findings at the site of infection. Results. Monocytes differentiated in presence of platelets (MP-Macs) acquired a foamy, epithelioid appearance and gave rise to MNGCs (MP-MNGCs). MP-Macs up-regulated activation markers, phagocytosed mycobacteria, and released abundant interleukin 10. Upon extended culture, MP-Macs shared transcriptional features with epithelioid cells and M2 macrophages and up-regulated CXCL5 transcripts. In line with this, CXCL5 concentrations were significantly increased in airways of active tuberculosis patients. The platelet-specific CD42b antigen was detected in MP-Macs, likewise in macrophages, MNGCs, and epithelioid cells within tuberculosis granulomas, along with the platelet aggregation-inducing factor PDPN. Conclusions. Platelets drive macrophage differentiation into MNGCs with characteristics of epithelioid, foam, and giant cells observed in tuberculosis granulomas. Our data define platelets as novel participants in tuberculosis pathogenesis.

Increased frequencies of pulmonary regulatory T-cells in latent Mycobacterium tuberculosis infection.

Regulation of specific immune responses following exposure to Mycobacterium tuberculosis in humans and the role of regulatory T (Treg) cells in the immune control of latent infection with M. tuberculosis are incompletely understood. Latent infection was assayed by an interferon-&ggr; release assay (IGRA) in healthcare workers regularly exposed to tuberculosis (TB) patients and in household TB contacts in Germany. Immunophenotypes of bronchoalveolar lavage (BAL) mononuclear cells and peripheral blood mononuclear cells (PBMCs) were analysed by fluorescence-activated cell sorting. All TB contacts with latent infection (n=15) had increased (p<0.0001) frequencies of CD4+ CD25+ CD127- Treg cells (median 2.12%, interquartile range (IQR) 1.63–3.01%) among BAL mononuclear cells compared with contacts (n=25) with negative IGRA results (median 0.68%, IQR 0.32–0.96%) No differences were seen when PBMC immunophenotypes of IGRA+ and IGRA- TB contacts were compared (IGRA+ median 9.6%, IQR 5.9–10.1%; IGRA- median 7.7%, IQR 4.6–11.3%; p=0.47). Five out of 25 contacts with negative blood IGRAs showed a positive IGRA from BAL cells, possibly indicating a limited local immune response. In Germany, latent infection with M. tuberculosis, as defined by a positive M. tuberculosis-specific IGRA response on cells from the peripheral blood, is characterised by an increased frequency of Treg cells in the BAL.