MicroRNAs correlate with bacillary index and genes associated to cell death processes in leprosy.

Mycobacterium leprae infects skin and peripheral nerves causing a broad of clinical forms. MicroRNAs (miRNAs) control immune mechanisms such as apoptosis, autophagy as well as to target genes leading to abnormal proliferation, metastasis, and invasion of cells. Herein we evaluated miRNAs expression for leprosy phenotypes in biopsies obtained from patients with and without reactions. We also correlated those miRNAs with both, bacillary index (BI) and genes involved in the micobacteria elimination process. Our results show a significant increase in the miR-125a-3p expression in paucibacillary (PB) patients vs multibacillary (MB) subjects (p = 0.007) and vs reversal reactions (RR) (p = 0.005), respectively. Likewise, there was a higher expression of miR-125a-3p in patients with erythema nodosum leprosum (ENL) vs MB without reactions (p = 0.002). Furthermore, there was a positive correlation between miR-125a-3p, miR-146b-5p and miR-132-5p expression and BI in patients with RR and ENL. These miRNAS were also correlated with genes such as ATG12 (miR-125a-3p), TNFRSF10A (miR-146b-5p), PARK2, CFLAR and STX7 (miR-132-5p). All together we underpin a role for these miRNAs in leprosy pathogenesis, implicating mechanisms such as apoptosis and autophagy in skin. The miR-125a-3p might have a distinct role associated with PB phenotype and ENL in MB patients.

Dapsone modulates lipopolysaccharide-activated bone marrow cells by inducing cell death and down-regulating tumor necrosis factor-α production.

Dapsone, an antibiotic, has been used to cure leprosy. It has been reported that dapsone has anti-inflammatory activity in hosts; however, the anti-inflammatory mechanism of dapsone has not been fully elucidated. The present study investigated the anti-inflammatory effects of dapsone on bone marrow cells (BMs), especially upon exposure to lipopolysaccharide (LPS). We treated BMs with LPS and dapsone, and the treated cells underwent cellular activity assay, flow cytometry analysis, cytokine production assessment, and reactive oxygen species assay. LPS distinctly activated BMs with several characteristics including high cellular activity, granulocyte changes, and tumor necrosis factor alpha (TNF-α) production increases. Interestingly, dapsone modulated the inflammatory cells, including granulocytes in LPS-treated BMs, by inducing cell death. While the percentage of Gr-1 positive cells was 57% in control cells, LPS increased that to 75%, and LPS plus dapsone decreased it to 64%. Furthermore, dapsone decreased the mitochondrial membrane potential of LPS-treated BMs. At a low concentration (25 µg/mL), dapsone significantly decreased the production of TNF-α in LPS-treated BMs by 54%. This study confirmed that dapsone has anti-inflammatory effects on LPS-mediated inflammation via modulation of the number and function of inflammatory cells, providing new and useful information for clinicians and researchers.

Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation.

During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5--two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators--during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1ß activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread.

Suppression of ROS generation by 4,4-diaminodiphenylsulfone in non-phagocytic human diploid fibroblasts.

The action mode of 4,4-diaminodiphenylsulfone (DDS) is still under debate, although it has long been used in treatment of several dermatologic diseases including Hansens disease. In this study, we tested the effect of DDS as an antioxidant on paraquat-induced oxidative stress in non-phagocytic human diploid fibroblasts (HDFs). Overall, preincubation of HDFs with DDS prevented the oxidative stress and the resulting cytotoxic damages caused by paraquat in these cells. The specific effects of DDS in paraquat-treated HDFs are summarized as follows: a) reducing the expression of NADPH oxidase 4 (NOX4) by inhibiting paraquat-induced activation of PKC; b) inhibiting paraquat-induced decreases in mitochondrial complex protein levels as well as in membrane potentials; c) consequently, inhibiting the generation of cytosolic and mitochondrial superoxide anions. Taken together, these findings suggest that DDS would suppress the radical generation in non-phagocytic HDFs during oxidative stress, and that DDS might have the extended potential to be used further in prevention of other oxidative stress-related pathologies.

Introduction to the review series Autophagy in Higher Eukaryotes--a matter of survival or death.

Single celled eukaryotes utilize autophagy (or self-consumption) to adapt to fluctuating energy sources in the environment. The identification in multicellular organisms of orthologs of autophagy-related yeast genes has led to some of the major advances in the molecular dissection of the pathway in the last decade. In higher eukaryotes, autophagy is much more than a 'stress response' pathway. The complexity of multicellular systems calls for greater sophistication and coordination not only in regulating the stress response but also in sustaining normal physiological functions and a homeostatic environment in the whole organism. The review series on 'Autophagy in Higher Eukaryotes--a matter of survival or death' in the current issue comprises a variety of perspectives on the role of autophagy in cell growth, survival and death, in neurodegeneration, tumor suppression and tumor progression. For example, Høyer-Hansen and Jäättellä cogitate on the emergence of autophagy as a target in cancer therapy. In addition, Sanjuan and Green examine its role in the defense against microbial pathogens and Sachdeva and Thompson offer an intriguing look at autophagy in the context of circadian clocks and diurnal rhythms. Presented below are some of the salient points from these perspectives.

M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells.

M. tuberculosis and M. leprae are considered to be prototypical intracellular pathogens that have evolved strategies to enable growth in the intracellular phagosomes. In contrast, we show that lysosomes rapidly fuse with the virulent M. tuberculosis- and M. leprae-containing phagosomes of human monocyte-derived dendritic cells and macrophages. After 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in nonapoptotic cells. Cytosolic entry is also observed for M. leprae but not for vaccine strains such as M. bovis BCG or in heat-killed mycobacteria and is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This may also reveal a mechanism for MHC-based antigen presentation that is lacking in current vaccine strains.

The proteasome function is required for Mycobacterium leprae-induced apoptosis and cytokine secretion.

Previous studies have demonstrated the importance of the ubiquitin-proteasome pathway in the immune response to bacterial pathogens. To investigate the role of this system in the context of leprosy, Mycobacterium leprae-stimulated peripheral blood mononuclear cells (PBMC) were treated with the proteasome inhibitor MG132 to assess the levels of apoptosis and cytokine secretion. The results showed that the inhibition of proteasome activity significantly reduced M. leprae-mediated cell death. In addition, MG132 treatment led to a significant decrease in M. leprae-induced TNF-alpha and IL-10 secretion. Together, these results suggest that modulations of the ubiquitin-proteasome pathway may participate in the human response to M. leprae.

Early development of the optic nerve in the turtle Mauremys leprosa.

We show the distribution of the neural and non-neural elements in the early development of the optic nerve in the freshwater turtle, Mauremys leprosa, using light and electron microscopy. The first optic axons invaded the ventral periphery of the optic stalk in close relationship to the radial neuroepithelial processes. Growth cones were thus exclusively located in the ventral margin. As development progressed, growth cones were present in ventral and dorsal regions, including the dorsal periphery, where they intermingled with mature axons. However, growth cones predominated in the ventral part and axonal profiles dorsally, reflecting a dorsal to ventral gradient of maturation. The size and morphology of growth cones depended on the developmental stage and the region of the optic nerve. At early stages, most growth cones were of irregular shape, showing abundant lamellipodia. At the following stages, they tended to be larger and more complex in the ventral third than in intermediate and dorsal portions, suggesting a differential behavior of the growth cones along the ventro-dorsal axis. The arrival of optic axons at the optic stalk involved the progressive transformation of neuroepithelial cells into glial cells. Simultaneously with the fiber invasion, an important number of cells died by apoptosis in the dorsal wall of the optic nerve. These findings are discussed in relation to the results described in the developing optic nerve of other vertebrates.

Developmental changes in the fibre population of the optic nerve follow an avian/mammalian-like pattern in the turtle Mauremys leprosa.

The changes in the axon and growth cone numbers in the optic nerve of the freshwater turtle Mauremys leprosa were studied by electron microscopy from the embryonic day 14 (E14) to E80, when the animals normally hatch, and from the first postnatal day (P0) to adulthood (5 years on). At E16, the first axons appeared in the optic nerve and were added slowly until E21. From E21, the fibre number increased rapidly, peaking at E34 (570,000 fibres). Thereafter, the axon number decreased sharply, and from E47 declined steadily until reaching the mature number (about 330,000). These observations indicated that during development of the retina there was an overproduction and later elimination of retinal ganglion cells. Growth cones were first observed in the optic nerve at as early as E16. Their number increased rapidly until E21 and continued to be high through E23 and E26. After E26, the number declined steeply and by E40 the optic nerve was devoid of growth cones. These results indicated that differentiation of the retinal ganglion cells occurred during the first half of the embryonic life. To examine the correlation between the loss of the fibres from the optic nerve and loss of the parent retinal ganglion cells, retinal sections were processed with the TUNEL technique. Apoptotic nuclei were detected in the ganglion cell layer throughout the period of loss of the optic fibres. Our results showed that the time course of the numbers of the fibres in the developing turtle optic nerve was similar to those found in birds and mammals.

Secretion of ATP-utilizing enzymes, nucleoside diphosphate kinase and ATPase, by Mycobacterium bovis BCG: sequestration of ATP from macrophage P2Z receptors?

Mycobacterium bovis BCG secretes two ATP-scavenging enzymes, nucleoside diphosphate kinase (Ndk) and ATPase, during growth in Middlebrook 7H9 medium. In synthetic Sauton medium without any protein supplements, there is less secretion of these two enzymes unless proteins such as bovine serum albumin (BSA), ovalbumin or extracts of macrophages are added to the medium. There is a gradient of activity among various proteins in triggering the induction of secretion of these two enzymes. Other mycobacteria, such as M. smegmatis, primarily secrete Ndk, while M. chelonae does not appear to secrete either of these two enzymes. Purification of the enzymes from the culture filtrate of 7H9-grown M. bovis BCG cells and determination of the N-terminal amino-acid sequence have demonstrated a high level of sequence identity of one of the ATPases with DnaK, a heat shock chaperone, of M. tuberculosis and M. leprae, while that of Ndk shows significant identity with the Ndk of Myxococcus xanthus. As both Ndk and ATPase use ATP as a substrate, the physiological significance of the secretion of these two ATP-utilizing enzymes was explored. External ATP is important in the activation of macrophage surface-associated P2Z receptors, whose activation has been postulated to allow phagosome-lysosome fusion and macrophage cell death. We demonstrate that the presence of the filtrate containing these enzymes prevents ATP-induced macrophage cell death, as measured by the release of an intracellular enzyme, lactate dehydrogenase. In vitro complexation studies with purified Ndk/ATPase and hyperproduced P2Z receptor protein will demonstrate whether these enzymes may be used by mycobacteria to sequester ATP from the macrophage P2Z receptors, thereby preventing phagosome-lysosome fusion or macrophage apoptotic death.