Lipoarabinomannan, and its related glycolipids, induce divergent and opposing immune responses to Mycobacterium tuberculosis depending on structural diversity and experimental variations

Exposure to Mycobacterium tuberculosis (Mtb) may lead to active or latent tuberculosis, or clearance of Mtb, depending essentially on the quality of the host's immune response. This response is initiated through the interaction of Mtb cell wall surface components, mostly glycolipids, with cells of the innate immune system, particularly macrophages (Mfs) and dendritic cells (DCs). The way Mfs and DC alter their cytokine secretome, activate or inhibit different microbicidal mechanisms and present antigens and consequently trigger the T cell-mediated immune response impacts the host immune response against Mtb. Lipoarabinomannan (LAM) is one of the major cell wall components of Mtb. Mannosyl-capped LAM (ManLAM), and its related cell wall-associated types of glycolipids/lipoglycans, namely phosphatidylinositol mannosides (PIMs) and lipomannan (LM), exhibit important and distinct immunomodulatory properties. The structure, internal heterogeneity and abundance of these molecules vary between Mtb strains exhibiting distinct degrees of virulence. Thus ManLAM, LM and PIMs may be considered crucial Mtb-associated virulence factors in the pathogenesis of tuberculosis. Of particular relevance for this review, there is controversy about the specific immunomodulatory properties of these distinct glycolipids, particularly when tested as purified molecules in vitro. In addition to the variability in the glycolipid composition conflicting reports may also result from differences in the protocols used for glycolipid isolation and for in vitro experiments including immune cell types and procedures to generate them. Understanding the immunomodulatory properties of these cell wall glycolipids, how they differ between distinct Mtb strains, and how they influence the degree of Mtb virulence, is of utmost relevance to understand how the host mounts a protective or otherwise pathologic immune response. This is essential for the design of preventive strategies against tuberculosis. Thus, since clarifying the controversy on this matter is crucial we here review, summarize and discuss reported data from in vitro stimulation with the three major Mtb complex cell wall glycolipids (ManLAM, PIMs and LM) in an attempt to conciliate the conflicting findings.info:eu-repo/semantics/publishedVersio

Ultrasensitive detection of lipoarabinomannan with plasmonic grating biosensors in clinical samples of HIV negative patients with tuberculosis.

BACKGROUND:Timely diagnosis of tuberculosis disease is critical for positive patient outcomes, yet potentially millions go undiagnosed or unreported each year. Sputum is widely used as the testing input, but limited by its complexity, heterogeneity, and sourcing problems. Finding methods to interrogate noninvasive, non-sputum clinical specimens is indispensable to improving access to tuberculosis diagnosis and care. In this work, economical plasmonic gratings were used to analyze tuberculosis biomarker lipoarabinomannan (LAM) from clinical urine samples by single molecule fluorescence assay (FLISA) and compared with gold standard sputum GeneXpert MTB/ RIF, culture, and reference ELISA testing results. METHODS AND FINDINGS:In this study, twenty sputum and urine sample sets were selected retrospectively from a repository of HIV-negative patient samples collected before initiation of anti-tuberculosis therapy. GeneXpert MTB/RIF and culture testing of patient sputum confirmed the presence or absence of pulmonary tuberculosis while all patient urines were reference ELISA LAM-negative. Plasmonic gratings produced by low-cost soft lithography were bound with anti-LAM capture antibody, incubated with patient urine samples, and biotinylated detection antibody. Fluorescently labeled streptavidin revealed single molecule emission by epifluorescence microscope. Using a 1 fg/mL baseline for limit of detection, single molecule FLISA demonstrated good qualitative agreement with gold standard tests on 19 of 20 patients, including accurately predicting the gold-standard-negative patients, while one gold-standard-positive patient produced no observable LAM in urine. CONCLUSIONS:Single molecule FLISA by plasmonic grating demonstrated the ability to quantify tuberculosis LAM from complex urine samples of patients from a high endemic setting with negligible interference from the complex media itself. Moreover, agreement with patient diagnoses by gold standard testing suggests that single molecule FLISA could be used as a highly sensitive test to diagnose tuberculosis noninvasively

A sensitive urinary lipoarabinomannan test for tuberculosis

CITATION: Hamasur, B., et al. 2015. A sensitive urinary lipoarabinomannan test for tuberculosis. PLoS ONE, 10(4):1-11, doi:10.1371/journal.pone.0123457.The original publication is available at http://journals.plos.org/plosoneWe have previously developed a diagnostic test for tuberculosis based on detection of mycobacterial lipoarabinomannan (LAM) in urine. The method depended on a laborious concentration step. We have now developed an easy to perform test based on a magnetic immunoassay platform, utilizing high avidity monoclonal antibodies for the detection of LAM in urine. With this method the analytical sensitivity of the assay was increased 50-100-fold compared to conventional ELISA. In a pilot study of HIV-negative patients with microbiologically verified TB (n=17) and healthy controls (n=22) the sensitivity of the test was 82% and the specificity 100%. This is in stark positive contrast to a range of studies using available commercial tests with polyclonal anti-LAM Abs where the sensitivity of the tests in HIVnegative TB patients was very low.http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0123457Publisher's versio

Divergent effects of mycobacterial cell wall glycolipids on maturation and function of human monocyte-derived dendritic cells.

BACKGROUND: Mycobacterium tuberculosis (Mtb) is able to evade the immune defenses and may persist for years, decades and even lifelong in the infected host. Mtb cell wall components may contribute to such persistence by modulating several pivotal types of immune cells. Dendritic cells (DCs) are the most potent antigen-presenting cells and hence play a crucial role in the initial immune response to infections by connecting the innate with the adaptive immune system. PRINCIPAL FINDINGS: We investigated the effects of two of the major mycobacterial cell wall-associated types of glycolipids, mannose-capped lipoarabinomannan (ManLAM) and phosphatidylinositol mannosides (PIMs) purified from the Mtb strains H37Rv and Mycobacterium bovis, on the maturation and cytokine profiles of immature human monocyte-derived DCs. ManLAM from Mtb H37Rv stimulated the release of pro-inflammatory cytokines TNF, IL-12, and IL-6 and expression of co-stimulatory (CD80, CD86) and antigen-presenting molecules (MHC class II). ManLAM from M. bovis also induced TNF, IL-12 and IL-6 but at significantly lower levels. Importantly, while ManLAM was found to augment LPS-induced DC maturation and pro-inflammatory cytokine production, addition of PIMs from both Mtb H37Rv and M. bovis strongly reduced this stimulatory effect. CONCLUSIONS: These results indicate that the mycobacterial cell wall contains macromolecules of glycolipid nature which are able to induce strong and divergent effects on human DCs; i.e while ManLAM is immune-stimulatory, PIMs act as powerful inhibitors of DC cytokine responses. Thus PIMs may be important Mtb-associated virulence factors contributing to the pathogenesis of tuberculosis disease. These findings may also aid in the understanding of some earlier conflicting reports on the immunomodulatory effects exerted by different ManLAM preparations

Kinetics of mAb binding to immobilized LAM at various mAb concentrations using Biacore.

<p>Kinetics of mAb binding to immobilized LAM at various mAb concentrations using Biacore.</p

ELISA titration curve of inhouse Anti-LAM mAbs 25 and 170 compared to the reference anti-LAM mAbs CSU-35 and CSU-40.

<p>ELISA titration curve of inhouse Anti-LAM mAbs 25 and 170 compared to the reference anti-LAM mAbs CSU-35 and CSU-40.</p

Effect of urine volume on detection limit of the Uri-TB-direct assay.

<p>* OD at absorbance 450 nm</p><p>Effect of urine volume on detection limit of the Uri-TB-direct assay.</p

Detection of LAM by the Uri-TB-direct test in urine of patients with microbiologically verified TB.

<p>Results in optical density (OD) at absorbance 450 n.</p><p>* Sputum samples were liquefied and decontaminated using N-acetyl-L-cysteine and NaOH (final concentration 1.5%). Portions of the decontaminated pellet were used for microscopy, culture and PCR. Smear microscopy was performed using auramine staining. For culture the material was inoculated into a Mycobacteria Growth Indicator Tube (MGIT) (Becton Dickinson, USA) and solid Lowenstein Jensen medium and incubated for up to 42 days. The Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland) was used for PCR.</p><p>** Not done</p><p>Detection of LAM by the Uri-TB-direct test in urine of patients with microbiologically verified TB.</p

Detection of LAM by the Uri-TB-direct test in urine of healthy controls.

<p>Results in optical density (OD) at absorbance 450 n.</p><p>Detection of LAM by the Uri-TB-direct test in urine of healthy controls.</p