Type 1 Reaction in Patients With Leprosy Corresponds to a Decrease in Proresolving Lipid Mediators and an Increase in Proinflammatory Lipid Mediators.

Background: Type 1 reaction (T1R) is an acute T-helper type 1 (Th1) inflammatory episode in patients with leprosy. While immunological responses associated with T1R have been investigated, the corresponding metabolic responses that could contribute to T1R pathology have received little attention. Methods: Metabolomics-based analyses of sera from 7 patients with and 9 without T1R were conducted via liquid chromatography-mass spectrometry. Serum metabolites present at levels that significantly differed (P < .05) with a log2 fold change of ≥ 1.0 between patient groups were interrogated against known metabolic pathways. The structural identification of targeted metabolites was confirmed and abundance changes validated by mass spectrometry and enzyme-linked immunoassay. Results: Forty metabolic pathways were perturbed in patients with T1R, with 71 dysregulated metabolites mapping to pathways for lipid mediators of inflammation. Of note was an increase in the abundance of the proinflammatory leukotriene B4 (LTB4) and a corresponding decrease in the level of proresolving resolvin D1 (RvD1). Also, levels of prostaglandin D2 (PGD2) and lipoxin A4 (LXA4) in patients with T1R were significantly increased, while the level of prostaglandin E2 (PGE2) was decreased. Conclusions: The dysregulation of metabolic pathways leading to abundance shifts between proinflammatory and proresolving lipid mediators provides a link between metabolic and cellular immune responses that result in the Th1-mediated pathology of T1R.

Variable ventilation improves pulmonary function and reduces lung damage without increasing bacterial translocation in a rat model of experimental pneumonia.

BACKGROUND: Variable ventilation has been shown to improve pulmonary function and reduce lung damage in different models of acute respiratory distress syndrome. Nevertheless, variable ventilation has not been tested during pneumonia. Theoretically, periodic increases in tidal volume (VT) and airway pressures might worsen the impairment of alveolar barrier function usually seen in pneumonia and could increase bacterial translocation into the bloodstream. We investigated the impact of variable ventilation on lung function and histologic damage, as well as markers of lung inflammation, epithelial and endothelial cell damage, and alveolar stress, and bacterial translocation in experimental pneumonia. METHODS: Thirty-two Wistar rats were randomly assigned to receive intratracheal of Pseudomonas aeruginosa (PA) or saline (SAL) (n = 16/group). After 24-h, animals were anesthetized and ventilated for 2 h with either conventional volume-controlled (VCV) or variable volume-controlled ventilation (VV), with mean VT = 6 mL/kg, PEEP = 5cmH2O, and FiO2 = 0.4. During VV, tidal volume varied randomly with a coefficient of variation of 30% and a Gaussian distribution. Additional animals assigned to receive either PA or SAL (n = 8/group) were not ventilated (NV) to serve as controls. RESULTS: In both SAL and PA, VV improved oxygenation and lung elastance compared to VCV. In SAL, VV decreased interleukin (IL)-6 expression compared to VCV (median [interquartile range]: 1.3 [0.3-2.3] vs. 5.3 [3.6-7.0]; p = 0.02) and increased surfactant protein-D expression compared to NV (2.5 [1.9-3.5] vs. 1.2 [0.8-1.2]; p = 0.0005). In PA, compared to VCV, VV reduced perivascular edema (2.5 [2.0-3.75] vs. 6.0 [4.5-6.0]; p < 0.0001), septum neutrophils (2.0 [1.0-4.0] vs. 5.0 [3.3-6.0]; p = 0.0008), necrotizing vasculitis (3.0 [2.0-5.5] vs. 6.0 [6.0-6.0]; p = 0.0003), and ultrastructural lung damage scores (16 [14-17] vs. 24 [14-27], p < 0.0001). Blood colony-forming-unit (CFU) counts were comparable (7 [0-28] vs. 6 [0-26], p = 0.77). Compared to NV, VCV, but not VV, increased expression amphiregulin, IL-6, and cytokine-induced neutrophil chemoattractant (CINC)-1 (2.1 [1.6-2.5] vs. 0.9 [0.7-1.2], p = 0.025; 12.3 [7.9-22.0] vs. 0.8 [0.6-1.9], p = 0.006; and 4.4 [2.9-5.6] vs. 0.9 [0.8-1.4], p = 0.003, respectively). Angiopoietin-2 expression was lower in VV compared to NV animals (0.5 [0.3-0.8] vs. 1.3 [1.0-1.5], p = 0.01). CONCLUSION: In this rat model of pneumonia, VV improved pulmonary function and reduced lung damage as compared to VCV, without increasing bacterial translocation.

Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

Polyunsaturated Fatty Acid-Derived Lipid Mediators as Potential Biomarkers for Leprosy Among Individuals with Asymptomatic Mycobacterium leprae Infection.

Intra-household contacts (HCs) of leprosy patients are at increased risk of infection by Mycobacterium leprae and about ∼5-10% will develop active disease. A prognostic tool to identify HCs with the greatest risk of progressing to active disease would enhance early leprosy diagnosis and optimize prophylactic intervention. Previous metabolomics studies suggest that host lipid mediators derived from ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) are potential biomarkers for leprosy. In this study, we investigated retrospective sera of leprosy HCs by liquid chromatography-mass spectrometry and enzyme-linked immunoassay to determine whether circulating levels of ω-3 and ω-6 PUFA metabolites were altered in HCs that developed leprosy (HCDL) in comparison to those that did not (HCNDL). Sera were collected from HCs at the time of index case diagnosis and before clinical signs/symptoms of leprosy. Our findings showed that HCDL sera exhibited a distinct metabolic profile in comparison to HCDNL. Specifically, arachidonic acid, leukotriene B4, 11-hydroxyeicosatetraenoic acid, prostaglandin D2, and lipoxin A4 were elevated in HCDL. In contrast, prostaglandin E2 levels were reduced in HCDL. The ω-3 PUFAs, docosahexaenoic acid, eicosapentaenoic acid, and the docosahexaenoic acid-derived resolvin D1 and maresin-1 were also elevated in HCDL individuals compared to HCNDL. Principal component analyses provided further evidence that lipid mediators could serve as an early biomarker for progression to active leprosy. A logistic model identified resolvin D1 and D2, and prostaglandin D2 as having the greatest potential for early detection of HCs that will manifest leprosy.

A pilot metabolomics study of tuberculosis immune reconstitution inflammatory syndrome.

BACKGROUND: Diagnosis of paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is challenging and new tools are needed for early diagnosis as well as to understand the biochemical events that underlie the pathology in TB-IRIS. METHODS: Plasma samples were obtained from participants from a randomized HIV/TB treatment strategy study (AIDS Clinical Trials Group [ACTG] A5221) with (n = 26) and without TB-IRIS (n = 22) for an untargeted metabolomics pilot study by liquid-chromatography mass spectrometry. The metabolic profile of these participants was compared at the study entry and as close to the diagnosis of TB-IRIS as possible (TB-IRIS window). Molecular features with p < 0.05 and log2 fold change ≥0.58 were submitted for pathway analysis through MetaboAnalyst. We also elucidated potential metabolic signatures for TB-IRIS using a LASSO regression model. RESULTS: At the study entry, we showed that the arachidonic acid and glycerophospholipid metabolism were altered in the TB-IRIS group. Sphingolipid and linoleic acid metabolism were the most affected pathways during the TB-IRIS window. LASSO modeling selected a set of 8 and 7 molecular features with the potential to predict TB-IRIS at study entry and during the TB-IRIS window, respectively. CONCLUSION: This study suggests that the use of plasma metabolites may distinguish HIV-TB patients with and without TB-IRIS.

Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp.

BACKGROUND: The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs). In the present study, nuclear magnetic resonance (NMR) was used to map the binding site(s) of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction. RESULTS: The capacity of a panel of 30 mer synthetic peptides covering the full length of Hlp to bind to heparin/heparan sulfate was analyzed by solid phase assays, NMR, and affinity chromatography. An additional active region between the residues Gly46 and Ala60 was defined at the N-terminal domain of Hlp, expanding the previously defined heparin-binding site between Thr31 and Phe50. Additionally, the C-terminus, rich in Lys residues, was confirmed as another heparan sulfate binding region. The amino acids in Hlp identified as mediators in the interaction with heparan sulfate were Arg, Val, Ile, Lys, Phe, and Thr. CONCLUSION: Our data indicate that Hlp interacts with heparan sulfate through two distinct regions of the protein. Both heparan sulfate-binding regions here defined are preserved in all mycobacterial Hlp homologues that have been sequenced, suggesting important but possibly divergent roles for this surface-exposed protein in both pathogenic and saprophic species.

Host Lipid Mediators in Leprosy: The Hypothesized Contributions to Pathogenesis.

The spectrum of clinical forms observed in leprosy and its pathogenesis are dictated by the host's immune response against Mycobacterium leprae, the etiological agent of leprosy. Previous results, based on metabolomics studies, demonstrated a strong relationship between clinical manifestations of leprosy and alterations in the metabolism of ω3 and ω6 polyunsaturated fatty acids (PUFAs), and the diverse set of lipid mediators derived from PUFAs. PUFA-derived lipid mediators provide multiple functions during acute inflammation, and some lipid mediators are able to induce both pro- and anti-inflammatory responses as determined by the cell surface receptors being expressed, as well as the cell type expressing the receptors. However, little is known about how these compounds influence cellular immune activities during chronic granulomatous infectious diseases, such as leprosy. Current evidence suggests that specialized pro-resolving lipid mediators (SPMs) are involved in the down-modulation of the innate and adaptive immune response against M. leprae and that alteration in the homeostasis of pro-inflammatory lipid mediators versus SPMs is associated with dramatic shifts in the pathogenesis of leprosy. In this review, we discuss the possible consequences and present new hypotheses for the involvement of ω3 and ω6 PUFA metabolism in the pathogenesis of leprosy. A specific emphasis is placed on developing models of lipid mediator interactions with the innate and adaptive immune responses and the influence of these interactions on the outcome of leprosy.

Heparin-binding hemagglutinin (HBHA) of Mycobacterium leprae is expressed during infection and enhances bacterial adherence to epithelial cells.

A heparin-binding hemagglutinin (HBHA) expressed on the surface of Mycobacterium tuberculosis is an antigenic protein that has been implicated in bacterial adherence to epithelial cells and systemic dissemination. In this study, the potential role of the Mycobacterium leprae HBHA (ML-HBHA) homologue in leprosy was investigated. Initially, the in vivo expression of HBHA and its association with the M. leprae cell envelope was confirmed by immunoblotting and proteomic analysis. Mycobacterium leprae recombinant HBHA (rML-HBHA) bound to a heparin-Sepharose column, and its capacity to act as an adhesin was demonstrated in experiments showing that the exogenous addition of the protein to latex beads or to M. leprae cells promotes a dramatic increase in association with epithelial cells. Finally, serum anti-HBHA immunoglobulin G levels were investigated in individuals infected with M. leprae. Altogether, our data indicate that HBHA is recognized during the course of bacterial infection in humans and may play a role in leprosy pathogenesis.

Equine protease inhibitor system as a marker for the diagnosis of chronic obstructive pulmonary disease (COPD)

The protease inhibitor system (PI) was investigated to ascertain if it can be used as a marker of chronic obstructive pulmonary disease (COPD) in thoroughbred horses. Serum samples were taken from healthy thoroughbreds (n = 13) and those diagnosed as having COPD (n = 24) or inflammatory airway disease (IAD, n = 38) as well as from 3,600 undiagnosed thoroughbred horses. PI allelic and genotypic frequencies were estimated using protein electrophoresis on starch and polyacrylamide gels. The four groups of horses showed high genotypic similarity and none of the observed alleles or genotypes of the equine PI system were found to be associated with COPD.