Maria Cristina Vidal Pessolani

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.

Modulation of lipid droplets by Mycobacterium leprae in Schwann cells: a putative mechanism for host lipid acquisition and bacterial survival in phagosomes

The predilection of Mycobacterium leprae (ML) for Schwann cells (SCs) leads to peripheral neuropathy, a major concern in leprosy. Highly infected SCs in lepromatous leprosy nerves show a foamy, lipid‐laden appearance; but the origin and nature of these lipids, as well as their role in leprosy, have remained unclear. The data presented show that ML has a pronounced effect on host‐cell lipid homeostasis through regulation of lipid droplet (lipid bodies, LD) biogenesis and intracellular distribution. Electron microscopy and immunohistochemical analysis of lepromatous leprosy nerves for adipose differentiation‐related protein expression, a classical LD marker, revealed accumulating LDs in close association to ML in infected SCs. The capacity of ML to induce LD formation was confirmed in in vitro studies with human SCs. Moreover, via confocal and live‐cell analysis, it was found that LDs are promptly recruited to bacterial phagosomes and that this process depends on cytoskeletal reorganization and PI3K signalling. ML‐induced LD biogenesis and recruitment were found to be independent of TLR2 bacterial sensing. Notably, LD recruitment impairment by cytoskeleton drugs decreased intracellular bacterial survival. Altogether, our data revealed SC lipid accumulation in ML‐containing phagosomes, which may represent a fundamental aspect of bacterial pathogenesis in the nerve.

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.

Postgenomic approach to identify novel Mycobacterium leprae antigens with potential to improve immunodiagnosis of infection.

ABSTRACT Early detection of Mycobacterium leprae infection is considered an important component of strategies aiming at reducing transmission of infection, but currently available diagnostic tools often lack sufficient sensitivity and specificity to reach this goal. Recent comparative genomics have revealed the presence of 165 M. leprae genes with no homologue in M. tuberculosis. We selected 17 of these genes for further study. All 17 genes were found to be expressed at the mRNA level in M. leprae from infected mice and from a multibacillary leprosy patient. Additional comparative genomic analyses of all currently available mycobacterial genome databases confirmed 12 candidate genes to be unique to M. leprae, whereas 5 genes had homologues in mycobacteria other than M. tuberculosis. Evaluation of the immunogenicity of all 17 recombinant proteins in PBMC from 127 Brazilians showed that five antigens (ML0576, ML1989, ML1990, ML2283, and ML2567) induced significant gamma interferon levels in paucibacillary leprosy patients, reactional leprosy patients, and exposed healthy controls but not in most multibacillary leprosy patients, tuberculosis patients, or endemic controls. Importantly, among exposed healthy controls 71% had no detectable immunoglobulin M antibodies to the M. leprae-specific PGL-I but responded to one or more M. leprae antigen(s). Collectively, the M. leprae proteins identified are expressed at the transcriptome level and can efficiently activate T cells of M. leprae-exposed individuals. These proteins may provide new tools to develop tests for specific diagnosis of M. leprae infection and may enhance our understanding of leprosy and its transmission.

TLR6-Driven Lipid Droplets in Mycobacterium leprae-Infected Schwann Cells: Immunoinflammatory Platforms Associated with Bacterial Persistence

The mechanisms responsible for nerve injury in leprosy need further elucidation. We recently demonstrated that the foamy phenotype of Mycobacterium leprae-infected Schwann cells (SCs) observed in nerves of multibacillary patients results from the capacity of M. leprae to induce and recruit lipid droplets (LDs; also known as lipid bodies) to bacterial-containing phagosomes. In this study, we analyzed the parameters that govern LD biogenesis by M. leprae in SCs and how this contributes to the innate immune response elicited by M. leprae. Our observations indicated that LD formation requires the uptake of live bacteria and depends on host cell cytoskeleton rearrangement and vesicular trafficking. TLR6 deletion, but not TLR2, completely abolished the induction of LDs by M. leprae, as well as inhibited the bacterial uptake in SCs. M. leprae-induced LD biogenesis correlated with increased PGE2 and IL-10 secretion, as well as reduced IL-12 and NO production in M. leprae-infected SCs. Analysis of nerves from lepromatous leprosy patients showed colocalization of M. leprae, LDs, and cyclooxygenase-2 in SCs, indicating that LDs are sites for PGE2 synthesis in vivo. LD biogenesis Inhibition by the fatty acid synthase inhibitor C-75 abolished the effect of M. leprae on SC production of immunoinflammatory mediators and enhanced the mycobacterial-killing ability of SCs. Altogether, our data indicated a critical role for TLR6-dependent signaling in M. leprae–SC interactions, favoring phagocytosis and subsequent signaling for induction of LD biogenesis in infected cells. Moreover, our observations reinforced the role of LDs favoring mycobacterial survival and persistence in the nerve. These findings give further support to a critical role for LDs in M. leprae pathogenesis in the nerve.

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.

A Postgenomic Approach to Identification of Mycobacterium leprae-Specific Peptides as T-Cell Reagents

ABSTRACT To identify Mycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure to M. leprae from exposure to Mycobacterium tuberculosis or to environmental mycobacteria or from immune responses followingMycobacterium bovis BCG vaccination, 15-mer synthetic peptides were synthesized based on data from the M. lepraegenome, each peptide containing three or more predicted HLA-DR binding motifs. Eighty-one peptides from 33 genes were tested for their ability to induce T-cell responses, using peripheral blood mononuclear cells (PBMC) from tuberculoid leprosy patients (n = 59) and healthy leprosy contacts (n = 53) from Brazil, Ethiopia, Nepal, and Pakistan and 20 United Kingdom blood bank donors. Gamma interferon (IFN-γ) secretion proved more sensitive for detection of PBMC responses to peptides than did lymphocyte proliferation. Many of the peptides giving the strongest responses in leprosy donors compared to subjects from the United Kingdom, where leprosy is not endemic, have identical, or almost identical, sequences in M. leprae and M. tuberculosis and would not be suitable as diagnostic tools. Most of the peptides recognized by United Kingdom donors showed promiscuous recognition by subjects expressing differing HLA-DR types. The majority of the novel T-cell epitopes identified came from proteins not previously recognized as immune targets, many of which are cytosolic enzymes. Fifteen of the tested peptides had ≥5 of 15 amino acid mismatches between the equivalent M. leprae and M. tuberculosissequences; of these, eight gave specificities of ≥90% (percentage of United Kingdom donors who were nonresponders for IFN-γ secretion), with sensitivities (percentage of responders) ranging from 19 to 47% for tuberculoid leprosy patients and 21 to 64% for healthy leprosy contacts. A pool of such peptides, formulated as a skin test reagent, could be used to monitor exposure to leprosy or as an aid to early diagnosis.

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.

Deciphering the proteomic profile of Mycobacterium leprae cell envelope

The complete sequence of the Mycobacterium leprae genome, an obligate intracellular pathogen, shows a dramatic reduction of functional genes, with a coding capacity of less than 50%. Despite this massive gene decay, the leprosy bacillus has managed to preserve a minimal gene set, most of it shared with Mycobacterium tuberculosis, allowing its survival in the host with ensuing pathological manifestations. Thus, the identification of proteins that are actually expressed in vivo by M. leprae is of high significance in understanding obligate, intracellular mycobacterial pathogenesis. In this study, a high‐throughput proteomic approach was undertaken resulting in the identification of 218 new M. leprae proteins. Of these, 60 were in the soluble/cytosol fraction, 98 in the membrane and 104 in the cell wall. Although several proteins were identified in more than one subcellular fraction, the majority were unique to one. As expected, a high percentage of these included enzymes responsible for lipid biosynthesis and degradation, biosynthesis of the major components of the mycobacterial cell envelope, proteins involved in transportation across lipid barriers, and lipoproteins and transmembrane proteins with unknown functions. The data presented in this study contribute to our understanding of the in vivo composition and physiology of the mycobacterial cell envelope, a compartment known to play a major role in bacterial pathogenesis.

Mycobacterium leprae intracellular survival relies on cholesterol accumulation in infected macrophages: a potential target for new drugs for leprosy treatment

We recently showed that Mycobacterium leprae (ML) is able to induce lipid droplet formation in infected macrophages. We herein confirm that cholesterol (Cho) is one of the host lipid molecules that accumulate in ML‐infected macrophages and investigate the effects of ML on cellular Cho metabolism responsible for its accumulation. The expression levels of LDL receptors (LDL‐R, CD36, SRA‐1, SR‐B1, and LRP‐1) and enzymes involved in Cho biosynthesis were investigated by qRT‐PCR and/or Western blot and shown to be higher in lepromatous leprosy (LL) tissues when compared to borderline tuberculoid (BT) lesions. Moreover, higher levels of the active form of the sterol regulatory element‐binding protein (SREBP) transcriptional factors, key regulators of the biosynthesis and uptake of cellular Cho, were found in LL skin biopsies. Functional in vitro assays confirmed the higher capacity of ML‐infected macrophages to synthesize Cho and sequester exogenous LDL‐Cho. Notably, Cho colocalized to ML‐containing phagosomes, and Cho metabolism impairment, through either de novo synthesis inhibition by statins or depletion of exogenous Cho, decreased intracellular bacterial survival. These findings highlight the importance of metabolic integration between the host and bacteria to leprosy pathophysiology, opening new avenues for novel therapeutic strategies to leprosy.