CD4+CD25+ T regs with acetylated FoxP3 are associated with immune suppression in human leprosy.

Leprosy is a chronic human disease that results from infection of Mycobacterium leprae. T reg cells have been shown to have important implications in various diseases. However, in leprosy, it is still unclear whether T regs can mediate immune suppression during progression of the disease. In the present study, we have proposed the putative mechanism leading to high proportion of T reg cells and investigated its significance in human leprosy. High levels of TGF-ß followed by adaptation of FoxP3(+) naive and memory (CD4(+)CD45RA(+)/RO(+)) T cells were observed as the principal underlying factors leading to higher generation of T reg cells during disease progression. Furthermore, TGF-ß was found to be associated with increased phosphorylation-mediated-nuclear-import of SMAD3 and NFAT towards BL/LL pole to facilitate FoxP3 expression in these cells, the same as justified after using nuclear inhibitors of SMAD3 (SIS3) and NFAT (cyclosporin A) in CD4(+)CD25(+) cells in the presence of TGF-ß and IL-2. Interestingly, low ubiquitination of FoxP3 in T reg cells of BL/LL patients was revealed to be a major driving force in conferring stability to FoxP3 which in turn is linked to suppressive potential of T regs. The present study has also pinpointed the presence of CD4(+)CD25(+)IL-10(+) sub class of T regs (Tr1) in leprosy.

Thalidomide modulates Mycobacterium leprae-induced NF-κB pathway and lower cytokine response.

It is widely accepted that tumor necrosis factor alpha (TNF-α) plays a critical role in the development of tissue and nerve damage in leprosy and during the reactional episodes of acute inflammation. Thalidomide (N-α-phthalimidoglutarimide), a drug used to treat leprosy reaction, modulates immune response, inhibits inflammation and NF-κB activity. Here we investigated whether thalidomide inhibits NF-κB activation induced by Mycobacterium leprae, p38 and ERK1/2 MAPK activation. EMSA and supershift assays were performed to investigate NF-κB activation in response to M. leprae and its modulation following in vitro treatment with thalidomide. Luciferase assay was assayed in transfected THP-1 cells to determine NF-κB transcriptional activity. Flow cytometry and immunofluorescence were used to investigate p65 accumulation in the nucleus. Immunoblotting was used to investigate p38 and ERK1/2 phosphorylation. Following activation of PBMC and monocytes with M. leprae, the formation and nuclear localization of NF-κB complexes composed mainly of p65/p50 and p50/p50 dimers was observed. Induction of NF-κB activation and DNA binding activity was inhibited by thalidomide. The drug also reduced M. leprae-induced TNF-α production and inhibited p38 and ERK1/2 activation. Definition of the activation mechanisms in cells stimulated with M. leprae can lead to the development of new therapy applications to modulate NF-κB activation and to control the inflammatory manifestations due to enhanced TNF-α response as observed in leprosy and in leprosy reactions.

Mycobacterium indicus pranii supernatant induces apoptotic cell death in mouse peritoneal macrophages in vitro.

Mycobacterium indicus pranii (MIP), also known as Mw, is a saprophytic, non-pathogenic strain of Mycobacterium and is commercially available as a heat-killed vaccine for leprosy and recently tuberculosis (TB) as part of MDT. In this study we provide evidence that cell-free supernatant collected from original MIP suspension induces rapid and enhanced apoptosis in mouse peritoneal macrophages in vitro. It is demonstrated that the MIP cell-free supernatant induced apoptosis is mitochondria-mediated and caspase independent and involves mitochondrial translocation of Bax and subsequent release of AIF and cytochrome c from the mitochondria. Experiments with pharmacological inhibitors suggest a possible role of PKC in mitochondria-mediated apoptosis of macrophages.

Mycobacterium leprae induces NF-kappaB-dependent transcription repression in human Schwann cells.

Mycobacterium leprae, the causative agent of leprosy, invades peripheral nerve Schwann cells, resulting in deformities associated with this disease. NF-kappaB is an important transcription factor involved in the regulation of host immune antimicrobial responses. We aimed in this work to investigate NF-kappaB signaling pathways in the human ST88-14 Schwannoma cell line infected with M. leprae. Gel shift and supershift assays indicate that two NF-kappaB dimers, p65/p50 and p50/p50, translocate to the nucleus in Schwann cells treated with lethally irradiated M. leprae. Consistent with p65/p50 and p50/p50 activation, we observed IkappaB-alpha degradation and reduction of p105 levels. The nuclear translocation of p50/p50 complex due to M. leprae treatment correlated with repression of NF-kappaB-driven transcription induced by TNF-alpha. Moreover, thalidomide inhibited p50 homodimer nuclear translocation induced by M. leprae and consequently rescues Schwann cells from NF-kappaB-dependent transcriptional repression. Here, we report for the first time that M. leprae induces NF-kappaB activation in Schwann cells and thalidomide is able to modulate this activation.

Insights into regulation of human Schwann cell proliferation by Erk1/2 via a MEK-independent and p56Lck-dependent pathway from leprosy bacilli.

Activation of extracellular signal-regulated kinase (Erk) 1/2, which plays a critical role in diverse cellular processes, including cell proliferation, is known to be mediated by the canonical Raf-mitogen-activated protein kinase kinase (MEK) kinase cascade. Alternative MEK-independent signaling pathways for Erk1/2 activation in mammalian cells are not known. During our studies of human primary Schwann cell response to long-term infection of Mycobacterium leprae, the causative organism of leprosy, we identified that intracellular M. leprae activated Erk1/2 directly by lymphoid cell kinase (p56Lck), a Src family member, by means of a PKCepsilon-dependent and MEK-independent signaling pathway. Activation of this signaling induced nuclear accumulation of cyclin D1, G1/S-phase progression, and continuous proliferation, but without transformation. Thus, our data reveal a previously unknown signaling mechanism of glial cell proliferation, which might play a role in dedifferentiation as well as nerve regeneration and degeneration. Our findings may also provide a potential mechanism by which an obligate intracellular bacterial pathogen like M. leprae subverts nervous system signaling to propagate its cellular niche for colonization and long-term bacterial survival.

Evidence that C-terminal non-kinase domain of Pbs2p has a role in high osmolarity-induced nuclear localization of Hog1p.

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus. In baker's yeast external high osmolarity activates high osmolarity glycerol (HOG) MAPK pathway which consists of two upstream branches (SHO1 and SLN1) and common downstream elements Pbs2p MAPKK and Hog1p MAPK. Activation of this pathway causes rapid nuclear accumulation of Hog1p, essentially leading to the expression of target genes. Previously we have isolated a PBS2 homologue (DPBS2) from osmo-tolerant and salt-tolerant yeast Debaryomyces hansenii that partially complemented pbs2 mutation in Saccharomyces cerevisiae. Here we show that by replacing C-terminal region of Dpbs2p with the homologous region of Pbs2p we could abrogate partial complementation exhibited by Dpbs2p and this was achieved due to increase in nuclear translocation of Hog1p. Thus, our result showed that in HOG pathway, MAPKK has important role in nuclear translocation of Hog1p.

The role of ligand in estrogen receptor regulation of gene expression.

We believe that steroid binding is not required for receptor binding to DNA, but instead induces a conformational change in the receptor domains involved in the protein-protein interactions proposed above. Data from Hansen and Gorski (1986), and more recent studies (M. Fritsch and J. Gorski, unpublished results) strongly suggest that the steroid binding domain when bound to estrogens undergoes a dramatic change in conformation characterized by a loss of hydrophobic surface. This marked change in the steroid binding domain probably affects the so-called dimerization region located in this domain and thus the interaction of receptor with nuclear proteins in vivo. In our working model, ER is bound to specific DNA sequences or response elements of a variety of genes with or without estrogen. Ligand binding induces conformational changes in the steroid binding and perhaps other domains of the receptor that in turn change receptor interaction with the transcriptional machinery. The nature of this change is not at all clear at present, and the possibility of enzymatic modification of receptor or associated transcription factors should not be excluded. Whatever the mechanism of receptor action on transcription, we expect it kinetically will be closely related to the occupancy of the receptor with estrogen. Finally, any model of ER interactions with target genes also needs to account for the drastic ligand effect on the extractability of all ER from the nucleus.

Translocation of zinc from vacuole to nucleus during yeast meiosis.

A novel staining procedure employing the UV fluorochrome DAPI (4',6-diamidino-2-phenylindole X 2HCl) and dithizone (diphenylthiocarbazone) was developed for microcytochemical determination of sites of zinc localization in Saccharomyces cerevisiae Hansen. In vegetative cells vacuolar polyphosphate bodies stained with dithizone, whereas in sporulating cells nucleoli and centriolar plaques were dithizone-positive. Hence, dithizone not only permitted localization of zinc but also indicated zinc translocation from vacuolar to nuclear compartments during differentiation from the vegetative to sporulated state.