Geraldo M. B. Pereira

Lipid droplet formation in leprosy: Toll-like receptor-regulated organelles involved in eicosanoid formation and Mycobacterium leprae pathogenesis

A hallmark of LL is the accumulation of Virchows foamy macrophages. However, the origin and nature of these lipids, as well as their function and contribution to leprosy disease, remain unclear. We herein show that macrophages present in LL dermal lesions are highly positive for ADRP, suggesting that their foamy aspect is at least in part derived from LD (also known as lipid bodies) accumulation induced during ML infection. Indeed, the capacity of ML to induce LD formation was confirmed in vivo via an experimental model of mouse pleurisy and in in vitro studies with human peripheral monocytes and murine peritoneal macrophages. Furthermore, infected cells were shown to propagate LD induction to uninfected, neighboring cells by generating a paracrine signal, for which TLR2 and TLR6 were demonstrated to be essential. However, TLR2 and TLR6 deletions affected LD formation in bacterium‐bearing cells only partially, suggesting the involvement of alternative receptors of the innate immune response besides TLR2/6 for ML recognition by macrophages. Finally, a direct correlation between LD formation and PGE2 production was observed, indicating that ML‐induced LDs constitute intracellular sites for eicosanoid synthesis and that foamy cells may be critical regulators in subverting the immune response in leprosy.

Identification of specific proteins and peptides in Mycobacterium leprae suitable for the selective diagnosis of leprosy

Diagnosis of leprosy is a major obstacle to disease control and has been compromised in the past due to the lack of specific reagents. We have used comparative genome analysis to identify genes that are specific to Mycobacterium leprae and tested both recombinant proteins and synthetic peptides from a subset of these for immunological reactivity. Four unique recombinant proteins (ML0008, ML0126, ML1057, and ML2567) and a panel of 58 peptides (15 and 9 mer) were tested for IFN-γ responses in PBMC from leprosy patients and contacts, tuberculosis patients, and endemic and nonendemic controls. The responses to the four recombinant proteins gave higher levels of IFN-γ production, but less specificity, than the peptides. Thirty-five peptides showed IFN-γ responses only in the paucibacillary leprosy and household contact groups, with no responses in the tuberculosis or endemic control groups. High frequencies of IFN-γ-producing CD4+ and CD8+ T cells specific for the 15- and 9-mer peptides were observed in the blood of a paucibacillary leprosy patient. 9-mer peptides preferentially activated CD8+ T cells, while the 15-mer peptides were efficient in inducing responses in both the CD4+ and CD8+ T cell subsets. Four of the six 9-mer peptides tested showed promising specificity, indicating that CD8+ T cell epitopes may also have diagnostic potential. Those peptides that provide specific responses in leprosy patients from an endemic setting could potentially be developed into a rapid diagnostic test for the early detection of M. leprae infection and epidemiological surveys of the incidence of leprosy, of which little is known.

Immunological Cytokine Correlates of Protective Immunity and Pathogenesis in Leprosy

The in vitro production of interferon (IFN)‐γ, interleukin (IL)‐5, tumour necrosis factor (TNF)‐α and IL‐10 by blood mononuclear cells in response to whole Mycobacterium leprae and polyclonal stimulii of 23 individuals, representing a variety of conditions in relation to exposure/susceptibility to M. leprae, was assayed. In most cases, healthy household contacts of newly diagnosed multibacillary leprosy patients, designated exposed household contacts (EC), showed low‐to‐undetectable in vitro IFN‐γ production in addition to substantial TNF‐α production in response to M. leprae. In contrast, peripheral blood mononuclear cells from previously exposed contacts (R) regarded as resistant‐to‐leprosy released low‐to‐moderate levels of IFN‐γ together with a mixed cytokine profile resembling a T helper (Th)0‐type response. TNF‐α/IL‐10 ratios in response to M. leprae and Concanavalin A were significantly higher in EC than in R contacts suggesting a role for the TNF‐α/IL‐10 ratio in restraining mycobacteria proliferation and spreading early in infection. The cytokine profiles of leprosy patients were taken as reference points. Post‐treatment lepromatous leprosy patients secreted relatively high levels of IL‐10 in response to M. leprae, whereas one self‐cured tuberculoid leprosy patient produced simultaneously high levels of IFN‐γ and TNF‐α. In addition, the quantitative changes in the cytokines released by peripheral blood mononuclear cells in EC contacts after Bacille Calmette‐Guérin (BCG) vaccination were investigated. Vaccination induced amplification of IFN‐γ production with a concomitant decrease in TNF‐α/IL‐10 ratios that resembled the cytokine pattern observed in R contacts. IFN‐γ production was observed in response to both a cross‐reactive antigen (Ag 85) and a M. leprae‐specific protein (MMP‐I), which attests to a BCG nonspecific stimulation of the immune system, thereby casting these antigens as likely candidates for inclusion in a subunit vaccine against leprosy. Finally, a model for protective × pathologic response to mycobacteria is presented.

New Biomarkers with Relevance to Leprosy Diagnosis Applicable in Areas Hyperendemic for Leprosy

Leprosy is not eradicable with currently available diagnostics or interventions, as evidenced by its stable incidence. Early diagnosis of Mycobacterium leprae infection should therefore be emphasized in leprosy research. It remains challenging to develop tests based on immunological biomarkers that distinguish individuals controlling bacterial replication from those developing disease. To identify biomarkers for field-applicable diagnostics, we determined cytokines/chemokines induced by M. leprae proteins in blood of leprosy patients and endemic controls (EC) from high leprosy-prevalence areas (Bangladesh, Brazil, Ethiopia) and from South Korea, where leprosy is not endemic anymore. M. leprae-sonicate–induced IFN-γ was similar for all groups, excluding M. leprae/IFN-γ as a diagnostic readout. By contrast, ML2478 and ML0840 induced high IFN-γ concentrations in Bangladeshi EC, which were completely absent for South Korean controls. Importantly, ML2478/IFN-γ could indicate distinct degrees of M. leprae exposure, and thereby the risk of infection and transmission, in different parts of Brazilian and Ethiopian cities. Notwithstanding these discriminatory responses, M. leprae proteins did not distinguish patients from EC in one leprosy-endemic area based on IFN-γ. Analyses of additional cytokines/chemokines showed that M. leprae and ML2478 induced significantly higher concentrations of MCP-1, MIP-1β, and IL-1β in patients compared with EC, whereas IFN-inducible protein-10, like IFN-γ, differed between EC from areas with dissimilar leprosy prevalence. This study identifies M. leprae-unique Ags, particularly ML2478, as biomarker tools to measure M. leprae exposure using IFN-γ or IFN-inducible protein-10, and also shows that MCP-1, MIP-1β, and IL-1β can potentially distinguish pathogenic immune responses from those induced during asymptomatic exposure to M. leprae.

Pathogen-Specific Epitopes as Epidemiological Tools for Defining the Magnitude of Mycobacterium leprae Transmission in Areas Endemic for Leprosy

During recent years, comparative genomic analysis has allowed the identification of Mycobacterium leprae-specific genes with potential application for the diagnosis of leprosy. In a previous study, 58 synthetic peptides derived from these sequences were tested for their ability to induce production of IFN-γ in PBMC from endemic controls (EC) with unknown exposure to M. leprae, household contacts of leprosy patients and patients, indicating the potential of these synthetic peptides for the diagnosis of sub- or preclinical forms of leprosy. In the present study, the patterns of IFN-γ release of the individuals exposed or non-exposed to M. leprae were compared using an Artificial Neural Network algorithm, and the most promising M. leprae peptides for the identification of exposed people were selected. This subset of M. leprae-specific peptides allowed the differentiation of groups of individuals from sites hyperendemic for leprosy versus those from areas with lower level detection rates. A progressive reduction in the IFN-γ levels in response to the peptides was seen when contacts of multibacillary (MB) patients were compared to other less exposed groups, suggesting a down modulation of IFN-γ production with an increase in bacillary load or exposure to M. leprae. The data generated indicate that an IFN-γ assay based on these peptides applied individually or as a pool can be used as a new tool for predicting the magnitude of M. leprae transmission in a given population.

ML1419c Peptide Immunization Induces Mycobacterium leprae-Specific HLA-A*0201–Restricted CTL In Vivo with Potential To Kill Live Mycobacteria

MHC class I-restricted CD8+ T cells play an important role in protective immunity against mycobacteria. Previously, we showed that p113-121, derived from Mycobacterium leprae protein ML1419c, induced significant IFN-γ production by CD8+ T cells in 90% of paucibacillary leprosy patients and in 80% of multibacillary patients’ contacts, demonstrating induction of M. leprae-specific CD8+ T cell immunity. In this work, we studied the in vivo role and functional profile of ML1419c p113-121–induced T cells in HLA-A*0201 transgenic mice. Immunization with 9mer or 30mer covering the p113-121 sequence combined with TLR9 agonist CpG induced HLA-A*0201–restricted, M. leprae-specific CD8+ T cells as visualized by p113-121/HLA-A*0201 tetramers. Most CD8+ T cells produced IFN-γ, but distinct IFN-γ+/TNF-α+ populations were detected simultaneously with significant secretion of CXCL10/IFN-γ—induced protein 10, CXCL9/MIG, and VEGF. Strikingly, peptide immunization also induced high ML1419c-specific IgG levels, strongly suggesting that peptide-specific CD8+ T cells provide help to B cells in vivo, as CD4+ T cells were undetectable. An additional important characteristic of p113-121–specific CD8+ T cells was their capacity for in vivo killing of p113-121–labeled, HLA-A*0201+ splenocytes. The cytotoxic function of p113-121/HLA-A*0201–specific CD8+ T cells extended into direct killing of splenocytes infected with live Mycobacterium smegmatis expressing ML1419c: both 9mer and 30mer induced CD8+ T cells that reduced the number of ML1419c-expressing mycobacteria by 95%, whereas no reduction occurred using wild-type M. smegmatis. These data, combined with previous observations in Brazilian cohorts, show that ML1419c p113-121 induces potent CD8+ T cells that provide protective immunity against M. leprae and B cell help for induction of specific IgG, suggesting its potential use in diagnostics and as a subunit (vaccine) for M. leprae infection.

Mycobacterium leprae virulence-associated peptides are indicators of exposure to M. leprae in Brazil, Ethiopia and Nepal

Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.

Clinical, immunological and histological aspects of an uncommon type II reaction in patients with lepromatous leprosy

This study reports three cases of an unusual leprotic reaction characterized by superficial bullous ulcerative cutaneous lesions associated with high fever, malaise and oedema in patients with leprosy. Two patients responded to thalidomide treatment, with regression of the symptoms and skin ulcers. The third patient responded to thalidomide plus prednisone. Analysis of the ulcerated skin lesions showed dermal oedema with mononuclear cell infiltrate enriched for γδ‐positive T lymphocytes and an increased number of Mycobaterium leprae bacilli within capillary endothelium. In contrast, γδ+ cells were decreased in or absent from the blood. Tumour necrosis factor‐α and interleukin‐6 were raised in the serum of the patients at the onset of the reaction. After the episode, cytokine levels and the percentage of γδ+ cells in the blood returned to normal. These cases characterize an uncommon leprotic reaction with clinical similarities to type II reaction and may indicate a significant role for γδ+ T cells in its pathogenesis.

Mycobacterium leprae downregulates the expression of PHEX in Schwann cells and osteoblasts

Neuropathy and bone deformities, lifelong sequelae of leprosy that persist after treatment, result in significant impairment to patients and compromise their social rehabilitation. Phosphate-regulating gene with homologies to endopeptidase on the X chromosome (PHEX) is a Zn-metalloendopeptidase, which is abundantly expressed in osteoblasts and many other cell types, such as Schwann cells, and has been implicated in phosphate metabolism and X-linked rickets. Here, we demonstrate that Mycobacterium leprae stimulation downregulates PHEX transcription and protein expression in a human schwannoma cell line (ST88-14) and human osteoblast lineage. Modulation of PHEX expression was observed to a lesser extent in cells stimulated with other species of mycobacteria, but was not observed in cultures treated with latex beads or with the facultative intracellular bacterium Salmonella typhimurium. Direct downregulation of PHEX by M. leprae could be involved in the bone resorption observed in leprosy patients. This is the first report to describe PHEX modulation by an infectious agent.

Comparative aspects of T cell activation in vivo following stimulation with anti-CD3 MAB, allogeneic cells and trypanosoma cruzi

The in vivo administration of the immunosuppressive drug, Cyclosporin A (CSA), has allowed us to define IL-2 dependent and IL-2 independent pathways of T cell activation in vivo. Thus, CSA inhibited T cell activation and the production of IL-2 mRNA in the draining lymph node (LN) population following footpad injection of anti-CD3 mAb. In contrast, even though CSA completely inhibited the induction of IL-2 mRNA in the draining LN following the injection of allogeneic cells, T cell activation proceeded normally. In the present study, we have analyzed the effects of CSA on the T cell activation induced in vivo by T. cruzi. BALB/c and C57BL/6 mice were injected subcutaneously in the footpad with irradiated, cultured T. cruzi trypomastigotes (CMTs, clone sylvio-X10/4). CSA was delivered to the mice via an osmotic pump, Alzet 2001 at a concentration of 35mg/Kg/day. The injection of CMTs resulted in a dose dependent activation of the draining LN population including an increase in the number of cells, an increase in cell size, induction of expression of the IL-2 receptor and other T cell activation antigens (Ly-6, CD28), induction of responsiveness to IL-2, and a vigorous proliferative response when the freshly explanted node was cultured for 18 h in vitro in the presence of 3H-TdR. CSA markedly inhibited all of these parameters of T cell activation. Thus, the early T cell activation response observed after injection of irradiated T. cruzi CMTs appears to be mediated by an IL-2 dependent, CSA sensitive T cell activation pathway.