Functional biomarker signatures of circulating T-cells and its association with distinct clinical status of leprosy patients and their respective household contacts.

BACKGROUND: Leprosy is a chronic infectious disease classified into two subgroups for therapeutic purposes: paucibacillary (PB) and multibacillary (MB), closely related to the host immune responses. In this context it is noteworthy looking for immunological biomarkers applicable as complementary diagnostic tools as well as a laboratorial strategy to follow-up leprosy household contacts. METHODS: The cross-sectional study enrolled 49 participants, including 19 patients and 30 healthy controls. Peripheral blood mononuclear cells (PBMC) were isolated and incubated in the presence of Mycobacterium leprae bacilli. The cells were prepared for surface (CD4+ and CD8+) and intracytoplasmic cytokine staining (IFN-γ, IL-4 and IL-10). Multiple comparisons amongst groups were carried out by ANOVA, Kruskal-Wallis, Student T or Mann-Whitney test. Comparative analysis of categorical variables was performed by Chi-square. Functional biomarker signature analysis was conducted using the global median values for each biomarker index as the cut-off edge to identify the proportion of subjects with high biomarker levels. RESULTS: The cytokine signature analysis demonstrated that leprosy patients presented a polyfunctional profile of T-cells subsets, with increased frequency of IFN-γ+ T-cell subsets along with IL-10+ and IL-4+ from CD4+ T-cells, as compared to health Controls (Venn diagram report). Moreover, statistical analysis was carried out using parametric or non-parametric variance analysis followed by pairwise multiple comparisons, according to the data normality distribution. L(PB) displayed a polyfunctional profile characterized by enhanced percentage of IFN-γ+, IL-10+ and IL-4+ produced by most T-cell subsets, as compared to L(MB) that presented a more restricted cytokine functional profile mediated by IL-10+ and IL-4+ T-cells with minor contribution of IFN-γ produced by CD4+ T-cells. Noteworthy was that HHC(MB) exhibited enhanced frequency of IFN-γ+ T-cells, contrasting with HHC(PB) that presented a cytokine profile limited to IL-10 and IL-4. CONCLUSIONS: Our data demonstrated that L(PB) displayed enhanced percentage of IFN-γ+, IL-10+ and IL-4+ as compared to L(MB) that presented functional profile mediated by IL-10+ and IL-4+ T-cells and HHC(MB) exhibited enhanced frequency of IFN-γ+ T-cells, contrasting with HHC(PB). Together, our findings provide additional immunological features associated with leprosy and household contacts. These data provide evidence that biomarkers of immune response can be useful complementary diagnostic/prognostic tools as well as insights that household contacts should be monitored to access putative subclinical infection.

Effect of dialyzable leukocyte extract, sodium butyrate, and valproic acid in the development of anergy in murine leprosy.

Background: Murine leprosy is a chronic granulomatous disease caused by Mycobacterium lepraemurium (MLM) in mice and rats. The disease evolves with the development of cellular anergy that impedes the production of interferon gamma (IFNγ), tumor necrosis factor-alpha (TNFα), and nitric oxide (NO) required to kill the microorganism. In this study we investigated whether histone deacetylase inhibitors (HDACi) (valproic acid and sodium butyrate [NaB]) and the immunomodulator transfer factor in dialyzable leukocyte extracts (DLE) can prevent anergy in murine leprosy. Methods: Five groups of six Balb/c mice were intraperitoneally inoculated with 2 × 107 MLM. Thirty-days post inoculation, treatment was started; one group received no treatment, one was treated with rifampicin-clofazimine (R-C), one with sodium valproate (VPA), one with NaB, and one with DLE. The animals were monitored for the evidence of disease for 96 days. After euthanasia, their spleens were removed and processed for histologic, bacteriologic, and cytokine studies. Results: R-C completely controlled the ongoing disease. DLE and NaB significantly reduced the development of lesions, including granuloma size and the number of bacilli; VPA was less effective. DLE, NaB, and VPA reverted the anergic condition in diverse grades and allowed the expression of IFNγ, TNFα, and inducible NO synthase, also in diverse grades. Conclusion: Anergy in leprosy and murine leprosy allows disease progression. In this study, anergy was prevented, in significant degree, by DLE (an immunomodulator) and NaB (HDACi). VPA was less effective. These results suggest potential beneficial effects of DLE and NaB in the ancillary treatment of leprosy.

Mycobacterium lepraemurium uses TLR-6 and MR, but not lipid rafts or DC-sign, to gain access into mouse macrophages.

OBJECTIVE/BACKGROUND: Mycobacterium lepraemurium (MLM), the etiologic agent of murine leprosy, is an intracellular parasite of macrophages; the mechanism used by this bacterium to enter macrophages is not known. The fate of the MLM phagosome inside macrophages is also unknown. This study was conducted to investigate how MLM enters macrophages and to define the maturation process of MLM phagosome inside macrophages. MATERIALS AND METHODS: Peritoneal macrophages were incubated in the presence of mannan-bovine serum albumin (BSA), and antibodies to known macrophage receptors, including, anti-FcγRIII/RII (anti-CD16/32), anti-CD35 (anti-CR1), anti-TLR2, anti-TLR4, anti-TLR6, anti-CD14, and anti-dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). Then, macrophages were challenged with Iris Fuchsia-stained MLM, at a multiplicity of infection of 50:1. The blocking effect of the antibodies (and mannan-BSA) used was analyzed using direct microscopy and flow cytometry. The maturation process of MLM phagosomes was visualized by their interaction with antibodies to Rab5, Rab7, proton ATPase, and cathepsin D, by confocal microscopy. RESULTS: Only mannan-BSA and anti-TLR6 antibody significantly blocked the entry of MLM into macrophages. None of the other antibodies, including that for DC-SIGN, meaningfully inhibited the endocytic process. We also found that MLM is a fusiogenic mycobacterium. This was deduced from the orderly association of MLM phagosomes with Rab5, Rab7, Proton ATPase, and lysosomes (cathepsin D). CONCLUSION: Fusion of MLM phagosomes with lysosomes seems to be a necessary event for the intracellular multiplication of MLM; similar to Mycobacterium leprae, this microorganism hardly grows on artificial, synthetic, bacteriologic media.

Integrative analyses of leprosy susceptibility genes indicate a common autoimmune profile.

BACKGROUND: Leprosy is an ancient chronic infection in the skin and peripheral nerves caused by Mycobacterium leprae. The development of leprosy depends on genetic background and the immune status of the host. However, there is no systematic view focusing on the biological pathways, interaction networks and overall expression pattern of leprosy-related immune and genetic factors. OBJECTIVES: To identify the hub genes in the center of leprosy genetic network and to provide an insight into immune and genetic factors contributing to leprosy. METHODS: We retrieved all reported leprosy-related genes and performed integrative analyses covering gene expression profiling, pathway analysis, protein-protein interaction network, and evolutionary analyses. RESULTS: A list of 123 differentially expressed leprosy related genes, which were enriched in activation and regulation of immune response, was obtained in our analyses. Cross-disorder analysis showed that the list of leprosy susceptibility genes was largely shared by typical autoimmune diseases such as lupus erythematosus and arthritis, suggesting that similar pathways might be affected in leprosy and autoimmune diseases. Protein-protein interaction (PPI) and positive selection analyses revealed a co-evolution network of leprosy risk genes. CONCLUSIONS: Our analyses showed that leprosy associated genes constituted a co-evolution network and might undergo positive selection driven by M. leprae. We suggested that leprosy may be a kind of autoimmune disease and the development of leprosy is a matter of defect or over-activation of body immunity.

Induction and treatment of anergy in murine leprosy.

Leprosy is a disease consisting of a spectrum of clinical, bacteriological, histopathological and immunological manifestations. Tuberculoid leprosy is frequently recognized as the benign polar form of the disease, while lepromatous leprosy is regarded as the malignant form. The different forms of leprosy depend on the genetic and immunological characteristics of the patient and on the characteristics of the leprosy bacillus. The malignant manifestations of lepromatous leprosy result from the mycobacterial-specific anergy that develops in this form of the disease. Using murine leprosy as a model of anergy in this study, we first induced the development of anergy to Mycobacterium lepraemurium (MLM) in mice and then attempted to reverse it by the administration of dialysable leucocyte extracts (DLE) prepared from healthy (HLT), BCG-inoculated and MLM-inoculated mice. Mice inoculated with either MLM or BCG developed a robust cell-mediated immune response (CMI) that was temporary in the MLM-inoculated group and long-lasting in the BCG-inoculated group. DLE were prepared from the spleens of MLM- and BCG-inoculated mice at the peak of CMI. Independent MLM intradermally-inoculated groups were treated every other day with HLT-DLE, BCG-DLE or MLM-DLE, and the effect was documented for 98 days. DLE administered at a dose of 1.0 U (1 × 10(6) splenocytes) did not affect the evolution of leprosy, while DLE given at a dose of 0.1 U showed beneficial effects regardless of the DLE source. The dose but not the specificity of DLE was the determining factor for reversing anergy.

The innate immune response in leprosy.

Investigation into the innate immune response in leprosy has provided insight into immunoregulation in human infectious disease. Key advances include the role of pattern recognition receptors in recognizing pathogen-associated molecular patterns of Mycobacterium leprae, cytokine release by innate immune cells, macrophage and dendritic cell differentiation, as well as antimicrobial effector pathways. These insights provide targets for therapeutic intervention in modulating the course of leprosy and other chronic infectious diseases.

[Serological and cellular reactivity to mycobacterial proteins in Hansen's disease]. / Reactividad serológica y celular frente a proteínas micobacterianas en la enfermedad de Hansen.

The study was designed for evaluating immunological reactivity to various mycobacterial protein preparations using serological and cell-mediated immunological tests in patients with clinical leprosy signs, predominantly, with the multibacillary forms. All patients were adults with ages between 20 and 30 years. Fifty eight (n = 81) percent corresponded to Lepromatous Leprosy (LL), 29% (n = 41) to Borderline Lepromatous Leprosy (BL) and 10% (n = 41) to Borderline Borderline Leprosy (BB); only 3% were Borderline Tuberculoid (BT) patients: 74% males and 26% females. The most frequent reactional phenomenon was of the Erythema Nodosum (ENL) type. The mycobacterial proteins tested were: total crude Mycobacterium leprae antigens (MISA); Mycobacterium bovis (MbSA and excretion MbSA); partially purified excretion protein antigen, with a 30 kDa relative movility (Ml30); and recombinant M. leprae proteins (Mt70, Mb 65, Ml 36, 28, 18 and 10 kDa). Two of the recombinant proteins (Ml10 and Ml 36 kDa) presented a statiscally significant higher serological reactivity, directly related with a larger bacillary load (p = 0.0051 and 0.050 respectively). The 30 kDa protein was predominantly recognized by antibodies from multibacillary patients. Results show that mean antibody values were higher in non reactional patients when tested against complete proteins (MbSA and ex MbSA) when compared with the group of patients who presented reactional phenomena (p = 0.000567 and 0.000061, respectively). Comparing reactional with non reactional patients, it was seen that mean antibody values against complete proteins (MbSA and ex MbSA) were higher in non reactional individuals (p = 0.000567 and 0.000061, respectively). This same behavior occurred towards individual mycobacterial proteins (30, 10 and 36 kDa). The T lymphocyte prolypherative response in reactional and non reactional patients towards mycobacterial proteins (MlSA, Ml 10 kDa, MbSA, ex MbSA) was negative.

Palsy of the rear limbs in Mycobacterium lepraemurium-infected mice results from bone damage and not from nerve involvement.

A small but relatively constant proportion (3-5%) of mice chronically infected with Mycobacterium lepraemurium (MLM) develops bilateral paralysis of the rear limbs. The aim of the study was to investigate whether or not the bilateral leg palsy results from nerve involvement. Direct bacterial nerve infection or acute/delayed inflammation might possibly affect the nerves. Therefore, palsied animals were investigated for the presence of: (a) histopathological changes in the leg tissues including nerves, bones and annexes, and (b) serum antibodies to M. lepraemurium and M. leprae lipids, including phenolic glycolipid I from M. leprae. Histopathological study of the palsied legs revealed that the paralysis was not the result of direct involvement of the limb nerves, as neither bacilli nor inflammatory cells were observed in the nerve branches studied. Antibodies to brain lipids and cardiolipin were not detected in the serum of the palsied animals, thus ruling out an immune response to self-lipids as the basis for the paralysis. Although high levels of antibodies to MLM lipids were detected in the serum of palsied animals they were not related to limb paralysis, as the nerves of the palsied legs showed no evidence of inflammatory damage. In fact, nerves showed no evidence of damage. Paralysis resulted from severe damage of the leg bones. Within the bones the bone marrow became replaced by extended bacilli-laden granulomas that frequently eroded the bone wall, altering the normal architecture of the bone and its annexes, namely muscle, tendons and connective tissue. Although this study rules out definitively the infectious or inflammatory damage of nerves in murine leprosy, it opens a new avenue of research into the factors that participate in the involvement or the sparing of nerves in human and murine leprosy, respectively.

Contrary to BCG, MLM fails to induce the production of TNF alpha and NO by macrophages.

Pathogenic mycobacteria must possess efficient survival mechanisms to resist the harsh conditions of the intraphagosomal milieu. In this sense, Mycobacterium lepraemurium (MLM) is one of the most evolved intracellular parasites of murine macrophages; this microorganism has developed a series of properties that allows it not only to resist, but also to multiply within the inhospitable environment of the phagolysosome. Inside the macrophages, MLM appears surrounded by a thick lipid-envelope that protects the microorganism from the digestive effect of the phagosomal hydrolases and the acid pH. MLM produces a disease in which the loss of specific cell-mediated immunity ensues, thus preventing activation of macrophages. In vitro, and possibly also in vivo, MLM infects macrophages without triggering the oxidative (respiratory burst) response of these cells, thus preventing the production of the toxic reactive oxygen intermediaries (ROI). Supporting the idea that MLM is within the most evolved pathogenic microorganisms, in the present study we found, that contrary to BCG, M. lepraemurium infects macrophages without stimulating these cells to produce meaningful levels of tumor necrosis factor alpha (TNF alpha) or nitric oxide (NO). Thus, the ability of the microorganisms to stimulate in their cellular hosts, the production of ROI and RNI (reactive nitrogen intermediates), seems to be an inverse correlate of their pathogenicity; the lesser their ability, the greater their pathogenicity.

Mycobacterium leprae and Mycobacterium lepraemurium infections in domestic and wild animals.

Mycobacterium leprae, the aetiological agent of leprosy in humans, gives rise to a chronic granulomatous disease that affects primarily the skin and peripheral nerves, and secondarily some internal organs such as the testis and the eye; viscera are seldom involved. Depending on host resistance, leprosy may present as a benign disease (tuberculoid leprosy) or as a malignant disease (lepromatous leprosy), with a spectrum of intermediate stages appearing between the two. Immunity against leprosy depends on the cell-mediated immunity of the host, and this is severely compromised in the malignant (lepromatous) form of leprosy. Although culture of M. leprae has never been achieved in artificial media, the bacterium may be grown in several experimental animals, including the armadillo, non-human primates, and to a certain extent, rodents. Naturally acquired leprosy has been reported in wild nine-banded armadillos (Dasypus novemcinctus) and in three species of non-human primates (chimpanzees [Pan troglodytes], sooty mangabey monkeys [Cercocebus atys] and cynomolgus macaques [Macaca fascicularis]), thus qualifying leprosy as a zoonosis. Murine leprosy is a leprosy-like disease of rats and mice, caused by Mycobacterium lepraemurium. The disease affects primarily viscera and the skin, and very rarely peripheral nerves. Depending on the host strain, rodent leprosy may also evolve as 'lepromatous' or 'tuberculoid' leprosy, and strains of mouse that develop intermediate forms of the disease may exist. Growth of M. lepraemurium on conventional media for mycobacteria is not successful, but the bacterium has been cultured on an egg yolk-based medium. Naturally acquired murine leprosy has been observed in rats, mice and cats, but not in humans or any other species. Thus, in contrast to human leprosy, murine leprosy is not a zoonosis.

Susceptibility of "et," the spontaneously mutating CD1-derived nude mouse, to infection of M. lepraemurium.

We have studied the susceptibility to infection by Mycobacterium lepraemurium (MLM) of a nude, hypothymic, CD1-derived, spontaneous mouse mutant called "et" because of its extraterrestrial appearance. We found that despite their hypothymia, et/et mice were not more susceptible to infection by MLM than their euthymic et/+ counterparts. Infection of both et/et and et/+ mice with 50 x 10(6) bacilli by the intraperitoneal route led only to a mild infection with low levels of antimycobacterial antibodies and a small number of lesions. These lesions were indicative of reactive hepatitis and hyaline perisplenitis with lymphoid hyperplasia. Some small bacilliferous granulomas were also observed at the end of the experiment (5 months of infection). CD1 mice behave in a rather "resistant" manner to the infection by MLM. It is clear that the nu gene is not necessarily linked to the thymus defect, and it is also clear that the hypothymia of et/et mice does not obviously affect their general cell-mediated immune competence.

Susceptibility of et, the spontaneously mutating CD-1 derived nude mouse, to infection of M. lepraemurium

We have studied the susceptibility to infection by Mycobacterium lepraemurium (MLM) of a nude, hypothymic, CD1-derived, spontaneous mouse mutant called [quot ]et[quot ] because of its extraterrestrial appearance. We found that despite their hypothymia, et/et mice were not more susceptible to infection by MLM than their euthymic et/+ counterparts. Infection of both et/et and et/+ mice with 50 x 10(6) bacilli by the intraperitoneal route led only to a mild infection with low levels of antimycobacterial antibodies and a small number of lesions. These lesions were indicative of reactive hepatitis and hyaline perisplenitis with lymphoid hyperplasia. Some small bacilliferous granulomas were also observed at the end of the experiment (5 months of infection). CD1 mice behave in a rather [quot ]resistant[quot ] manner to the infection by MLM. It is clear that the nu gene is not necessarily linked to the thymus defect, and it is also clear that the hypothymia of et/et mice does not obviously affect their general cell-mediated immune competence.

Do antibodies to phospholipid antigens play any role in murine leprosy?

In order to know whether antibodies to phospholipids and other host lipids play a role in the pathology of murine leprosy, we looked for the presence of antibodies to cardiolipin, cerebroside sulfatide, and to lipids extracted from normal murine spleen, liver and brain in the sera of mice bearing a 6-month infection with Mycobacterium lepraemurium. We also looked for the presence of antibodies to lipids isolated from M. lepraemurium. We found that all of the 16 animals examined contained high levels of antibodies to the mycobacterial lipids of intermediate polarity (mostly glycolipids) but none of them had antibodies to the other lipids tested, including those isolated from mouse liver, spleen and brain, bovine cardiolipin and sulfatide, nor any significant levels of antibodies to mycobacterial lipids of high or low polarity. The infected animals also had high levels of antibodies to antigens sonically extracted from the microorganism. Antibodies to the socially extracted antigens (mostly proteins) were mainly IgG, while antibodies to the lipid antigens were predominantly IgM. Despite the low but significant percentage (1%-3%) of infected animals developing bilateral paralysis of the rear limbs, autoimmunity (due to antibodies to phospholipids and other host lipids) does not seem to be a feature of murine leprosy.

Recognition of lipid antigens by sera of mice infected with Mycobacterium lepraemurium.

Lipids extracted from mouse tissues infected with Mycobacterium lepraemurium (MLM) were analyzed by thin-layer chromatography. Although the extracted lipids were heterogeneous in polarity, the lipids of intermediate polarity were the ones that predominated. All of the lipids of intermediate polarity were glycosylated species. There were also lipids of low and high polarity, the latter being glycolipids. Compared to lipids extracted from normal tissue (mostly to lipids of high and low polarity), all of the additional lipids extracted from the infected tissue corresponded to lipids present in the purified bacteria. Enzyme-linked immunoassays (ELISAs) were then performed with the whole lipids extracted from purified bacilli, the lipids of high, intermediate and low polarity, and the sera from 20 normal and 20 MLM-infected mice. Lipids of intermediate polarity were specifically recognized by MLM-infected mice. Neither sera (diluted 1:500) from normal mice nor infected mice reacted with the lipids of high or low polarity, but a higher concentration (sera diluted 1:100) of some sera from mice in both groups reacted significantly with these lipids. In the ELISAs the whole-lipid extract and the lipids of intermediate polarity were similarly recognized by the sera of the infected mice. Thus, as observed in human leprosy, the mycobacterial disease in the mouse (murine leprosy) is also accompanied by the development of antibodies to the glycolipids of the infecting microorganism.

[BCG vaccination to Mycobacterium leprae infection in mice].

BCG vaccine (Tokyo strain) was given in BALB/cA mice intradermally 1 or 3 months before Mycobacterium leprae (M. leprae) challenge as modified Shepard's method. The vaccine dosage was 10(7-8) or 10(6). The BCG gave good protection in both dosages and both challenges against M. leprae infection. Lymphocytes proliferations of BCG-vaccinated splenocyte cultures in response to M. leprae lysate or BCG components (hsp65, 38 kD, 30 kD or 12 kD protein) were tested, and potent proliferative responses were seen in the cultures with M. leprae lysate and hsp65. Furthermore, gamma-IFN productions were positive in the cultures with M. leprae lysate or hsp65, but negative with other antigens. The production of gamma-IFN with hsp65 was never inhibited with polymyxin B, but inhibited with IL-10. These results show that BCG (Tokyo strain) is a useful vaccine for M. leprae infection in mice, and one of the components of BCG, hsp65, may be a effective antigen component for protection of M. leprae infection inducing Th1 type cytokine.

[The identification of the antigenic determinants of Mycobacterium leprae passed through laboratory animals by using monoclonal antibodies]. / Identifikatsiia antigennykh determinant Mycobacterium leprae, passiruemykh na laboratornykh zhivotnykh, s pomoshch'iu monoklonal'nykh antitel.

The antigenic preparations of M. leprae isolated from the biopsy leproma material of leprosy patients, M. leprae obtained by passage through laboratory animals (armadillos, rats) and M. lepraemurium have been identified by indirect solid-phase enzyme immunoassay. Experiments made with the use of the set of monoclonal antibodies (McAb) to different M. leprae antigenic determinants, supplied by the WHO Bank of Monoclonal Antibodies (IMMLEP), have demonstrated the presence of common antigenic determinants in 65-kD protein of M. leprae obtained by passage through armadillos and rats and M. lepraemurium, as well as in 12-kD protein of M. leprae isolated from armadillos and rats and M. leprae isolated from the biotic material of human lepromas. The passage of M. leprae through experimental animals leads to the appearance of a new antigenic determinant in their structure, which is recognized by McAb 111E4.

Inhibition of complement activity in murine leprosy.

NIH mice infected with Mycobacterium lepraemurium (MLM) show a marked depression in their levels of hemolytic complement that is proportional to the degree of infection. The defect affects more the activation of complement through the classical pathway (CPW) than the activation of complement through the alternative pathway. Although this low activity of CPW-complement may be due to different causes (complement consumption by the infecting microorganism, lack of biosynthesis of complement components, or the presence of complement inhibitory factors), our results seem to support the last possibility. The generation of factors in the infected animals that inhibit the autologous activity of complement as the infection goes on reduces the risk of complement-mediated tissue damage and prolongs the survival time of the host, a wise strategy on the part of the MLM to assure its own survival as a parasite.

Activated murine natural killer cells control growth of Mycobacterium lepraemurium in mouse macrophages; in vitro and in vivo evidence.

The role of natural killer cells (NK) in murine leprosy was investigated in vivo and in vitro. In a first set of experiments, it was found that IL-2 (interleukin-2) activated NK cells reduced Mycobacterium lepraemurium (MLM) growth in mouse C57BL/J peritoneal macrophages which had phagocytosed low numbers (MOI of 10 : 1) of MLM (P less than 0.0001 at day 20). There was no cytotoxicity exerted by the NK cells against the infected cells in these conditions. Conversely, macrophages heavily infected with MLM (multiplicity of infection of 1000 : 1) were found to be susceptible to lysis by activated NK cells in vitro. In vivo, progressing murine leprosy was associated with a sharp increase in splenic NK cell activity, which was abrogated by treatment with a monoclonal antibody against NK cells. Administration of this monoclonal antibody against NK cells enhanced C57BL6/J mouse susceptibility to mouse leprosy, as seen by a decrease in survival time of mice infected with 10(7) MLM i.v. (81 days vs 110 days, P less than 0.0005). Overall, these findings suggest that NK cells may play an important role in resistance to leprosy, either by reducing MLM growth in macrophages or by lysing heavily infected macrophages.