Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice.

Introduction: The large family of PE and PPE proteins accounts for as much as 10% of the genome of Mycobacterium tuberculosis. In this study, we explored the immunogenicity of three proteins from this family, PE18, PE31, and PPE26, in humans and mice. Methods: The investigation involved analyzing the immunoreactivity of the selected proteins using sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy donors from the TB endemic country Mozambique. Antigen-recall responses were examined in PBMC from these groups, including the evaluation of cellular responses in healthy unexposed individuals. Moreover, systemic priming and intranasal boosting with each protein, combined with the Quil-A adjuvant, were conducted in mice. Results: We found that all three proteins are immunoreactive with sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy controls. Likewise, antigen-recall responses were induced in PBMC from all groups, and the proteins stimulated proliferation of peripheral blood mononuclear cells from healthy unexposed individuals. In mice, all three antigens induced IgG antibody responses in sera and predominantly IgG, rather than IgA, responses in bronchoalveolar lavage. Additionally, CD4+ and CD8+ effector memory T cell responses were observed in the spleen, with PE18 demonstrating the ability to induce tissue-resident memory T cells in the lungs. Discussion: Having demonstrated immunogenicity in both humans and mice, the protective capacity of these antigens was evaluated by challenging immunized mice with low-dose aerosol of Mycobacterium tuberculosis H37Rv. The in vitro Mycobacterial Growth Inhibition Assay (MGIA) and assessment of viable bacteria in the lung did not demonstrate any ability of the vaccination protocol to reduce bacterial growth. We therefore concluded that these three specific PE/PPE proteins, while immunogenic in both humans and mice, were unable to confer protective immunity under these conditions.

Serological analysis reveals differential antibody responses between TB patients and latently infected individuals from the TB endemic country of Mozambique.

Serological antibody profiling of tuberculosis (TB) patients and household contacts with latent TB infection (LTBI) could identify risk indicators of disease progression, and potentially also serve as an easily accessible diagnostic tool to discriminate between these two stages of Mycobacterium tuberculosis (Mtb) infection. Yet, despite significant efforts over many decades, neither application has yet fully materialised, and this is at least in part due to inconsistent and varying antibody profiles from different TB endemic regions. In this study, we conducted a retrospective exploratory analysis of serum antibodies in a cohort of active TB patients (ATB) and their interferon-gamma release assay (IGRA) positive household contacts (LTBI), as well as healthy controls (HC) from Mozambique, a country with a high TB burden from the Sub-Saharan region. Using several Mtb antigens as well as crude preparations of culture filtrate proteins (CFP) from Mtb and Bacille Calmette Guérin (BCG), we report that the most discriminatory response for TB and LTBI was observed for serum IgA antibodies to the MPT64 antigen, followed by IgG antibodies to Ag85B and CFP, with ATB patients having significantly higher levels than LTBI or BCG-vaccinated healthy controls. Conversely, sera from LTBI individuals had higher levels of IgG antibodies to the HBHA antigen than ATB. While our sample size (n = 21 for ATB, 18 for LTBI and 17 for HC) was too small to fully evaluate the diagnostic potential of these differing serological profiles, our study however preliminarily indicated high level of sensitivity (95%) and specificity (97%) of an ELISA MPT64-IgA test for discriminating TB from LTBI and healthy controls, supporting the notion that it alone, or possibly in combination with other antigens such as Ag85B or CFP could lead to development of an easily accessible diagnostic tool for TB.

Mycobacterium tuberculosis PE/PPE proteins enhance the production of reactive oxygen species and formation of neutrophil extracellular traps.

Introduction: Neutrophil granulocytes predominate in the lungs of patients infected with Mycobacterium tuberculosis (Mtb) in earlier stages of the disease. During infection, neutrophils release neutrophil extracellular traps (NETs), an antimicrobial mechanism by which a DNA-backbone spiked with antimicrobial components traps the mycobacteria. However, the specific mycobacterial factors driving NET formation remain unclear. Proteins from the proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family are critical to Mtb pathophysiology and virulence. Methods: Here, we investigated NET induction by PE18, PPE26, and PE31 in primary human blood-derived neutrophils. Neutrophils were stimulated with the respective proteins for 3h, and NET formation was subsequently assessed using confocal fluorescence microscopy. Intracellular ROS levels and cell necrosis were estimated by flow cytometry. Additionally, the influence of phorbol-12-myristate-13-acetate (PMA), a known NADPH oxidase enhancer, on NET formation was examined. Neutrophil integrity following incubation with the PE/PPE proteins was evaluated using transmission electron microscopy. Results: For the first time, we report that stimulation of primary human blood-derived neutrophils with Mtb proteins PE18, PPE26, and PE31 resulted in the formation of NETs, which correlated with an increase in intracellular ROS levels. Notably, the presence of PMA further amplified this effect. Following incubation with the PE/PPE proteins, neutrophils were found to remain viable and structurally intact, as verified through transmission electron microscopy, indicating the occurrence of vital NET formation. Discussion: These findings offer valuable insights that contribute to a better understanding of host-pathogen interactions during Mtb infection. Moreover, they underscore the significance of these particular Mtb antigens in triggering NET formation, representing a distinctive and previously unrecognized function of PE/PPE antigens.

Role of phagocyte extracellular traps during Mycobacterium tuberculosis infections and tuberculosis disease processes.

Mycobacterium tuberculosis (M.tb) infections remain one of the most significant causes of mortality worldwide. The current situation shows an emergence of new antibiotic-resistant strains making it difficult to control the tuberculosis (TB) disease. A large part of its success as a pathogen is due to its ability to persist for years or even decades without causing evident clinical manifestations. M.tb is highly successful in evading the host-defense by manipulating host-signalling pathways. Although macrophages are generally viewed as the key cell type involved in harboring M.tb, growing evidence shows that neutrophils also play a fundamental role. Both cells are known to act in multiple ways when encountering an invading pathogen, including phagocytosis, release of cytokines and chemokines, and oxidative burst. In addition, the formation of neutrophil extracellular traps (NETs) and macrophage extracellular traps (METs) has been described to contribute to M.tb infections. NETs/METs are extracellular DNA fibers with associated granule components, which are released upon activation of the cells by the pathogen or by pro-inflammatory mediators. On one hand, they can lead to a protective immune response by entrapment and killing of pathogens. However, on the other hand, they can also play a severe pathological role by inducing tissue damage. Extracellular traps (ETs) produced in the pulmonary alveoli can expand easily and expose tissue-damaging factors with detrimental effects. Since host-directed therapies offer a complementary strategy in TB, the knowledge of NET/MET formation is important for understanding potential protective versus detrimental pathways during innate immune signaling. In this review, we summarize the progress made in understanding the role of NETs/METs in the pathogenesis of TB.

Cellular and Molecular Alterations Underlying Abnormal Bone Growth in X-Linked Hypophosphatemia.

X-linked hypophosphatemia (XLH), the most common form of hereditary hypophosphatemic rickets, is caused by inactivating mutations of the phosphate-regulating endopeptidase gene (PHEX). XLH is mainly characterized by short stature, bone deformities and rickets, while in hypophosphatemia, normal or low vitamin D levels and low renal phosphate reabsorption are the principal biochemical aspects. The cause of growth impairment in patients with XLH is not completely understood yet, thus making the study of the growth plate (GP) alterations necessary. New treatment strategies targeting FGF23 have shown promising results in normalizing the growth velocity and improving the skeletal effects of XLH patients. However, further studies are necessary to evaluate how this treatment affects the GP as well as its long-term effects and the impact on adult height.

Innovative Three-Dimensional Microscopic Analysis of Uremic Growth Plate Discloses Alterations in the Process of Chondrocyte Hypertrophy: Effects of Growth Hormone Treatment.

Chronic kidney disease (CKD) alters the morphology and function of the growth plate (GP) of long bones by disturbing chondrocyte maturation. GP chondrocytes were analyzed in growth-retarded young rats with CKD induced by adenine intake (AD), control rats fed ad libitum (C) or pair-fed with the AD group (PF), and CKD rats treated with growth hormone (ADGH). In order to study the alterations in the process of GP maturation, we applied a procedure recently described by our group to obtain high-quality three-dimensional images of whole chondrocytes that can be used to analyze quantitative parameters like cytoplasm density, cell volume, and shape. The final chondrocyte volume was found to be decreased in AD rats, but GH treatment was able to normalize it. The pattern of variation in the cell cytoplasm density suggests that uremia could be causing a delay to the beginning of the chondrocyte hypertrophy process. Growth hormone treatment appears to be able to compensate for this disturbance by triggering an early chondrocyte enlargement that may be mediated by Nkcc1 action, an important membrane cotransporter in the GP chondrocyte enlargement.

A simple method based on confocal microscopy and thick sections recognizes seven subphases in growth plate chondrocytes.

This manuscript reports a novel procedure to imaging growth plate chondrocytes by using confocal microscopy. The method is based on fixed undecalcified bone samples, in-block staining with eosin, epoxy resin embedding and grinding to obtain thick sections. It is simple, inexpensive and provides three-dimensional images of entire chondrocytes inside their native lacunae. Quantitative analysis of volume, shape and cytoplasm density of chondrocytes at different strata of the growth plate allowed to objectively grade chondrocytes of the growth plate in seven different clusters. These seven categories of chondrocytes were subsequently evaluated by immunohistochemistry of some well-defined molecular landmarks of chondrocyte differentiation. Furthermore, immunohistochemical analysis of proteins responsible for ionic changes and water transport allowing chondrocyte swelling during hypertrophy was also performed. Results obtained indicate that four subphases can be defined in the pre-hypertrophic zone and three subphases in the hypertrophic zone, a fact that raises that chondrocytes of the growth plate are less homogeneous than usually considered when different zones are defined according to subjective cell morphological criteria. Results in the present study provide a technological innovation and gives new insights into the complexity of the process of chondrocyte differentiation in the growth plate.