Genetic and functional analysis of common MRC1 exon 7 polymorphisms in leprosy susceptibility.

The chromosomal region 10p13 has been linked to paucibacillary leprosy in two independent studies. The MRC1 gene, encoding the human mannose receptor (MR), is located in the 10p13 region and non-synonymous SNPs in exon 7 of the gene have been suggested as leprosy susceptibility factors. We determined that G396S is the only non-synonymous exon 7-encoded polymorphism in 396 unrelated Vietnamese subjects. This SNP was genotyped in 490 simplex and 90 multiplex leprosy families comprising 704 patients (47% paucibacillary; 53% multibacillary). We observed significant under-transmission of the serine allele of the G396S polymorphism with leprosy per se (P = 0.036) and multibacillary leprosy (P = 0.034). In a sample of 384 Brazilian leprosy cases (51% paucibacillary; 49% multibacillary) and 399 healthy controls, we observed significant association of the glycine allele of the G396S polymorphism with leprosy per se (P = 0.016) and multibacillary leprosy (P = 0.023). In addition, we observed a significant association of exon 7 encoded amino acid haplotypes with leprosy per se (P = 0.012) and multibacillary leprosy (P = 0.004). Next, we tested HEK293 cells over-expressing MR constructs (293-MR) with three exon 7 haplotypes of MRC1 for their ability to bind and internalize ovalbumin and zymosan, two classical MR ligands. No difference in uptake was measured between the variants. In addition, 293-MR failed to bind and internalize viable Mycobacterium leprae and BCG. We propose that the MR-M. leprae interaction is modulated by an accessory host molecule of unknown identity.

TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells.

Leprosy enables investigation of mechanisms by which the innate immune system contributes to host defense against infection, because in one form, the disease progresses, and in the other, the infection is limited. We report that Toll-like receptor (TLR) activation of human monocytes induces rapid differentiation into two distinct subsets: DC-SIGN+ CD16+ macrophages and CD1b+ DC-SIGN- dendritic cells. DC-SIGN+ phagocytic macrophages were expanded by TLR-mediated upregulation of interleukin (IL)-15 and IL-15 receptor. CD1b+ dendritic cells were expanded by TLR-mediated upregulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor, promoted T cell activation and secreted proinflammatory cytokines. Whereas DC-SIGN+ macrophages were detected in lesions and after TLR activation in all leprosy patients, CD1b+ dendritic cells were not detected in lesions or after TLR activation of peripheral monocytes in individuals with the progressive lepromatous form, except during reversal reactions in which bacilli were cleared by T helper type 1 (TH1) responses. In tuberculoid lepromatous lesions, DC-SIGN+ cells were positive for macrophage markers, but negative for dendritic cell markers. Thus, TLR-induced differentiation of monocytes into either macrophages or dendritic cells seems to crucially influence effective host defenses in human infectious disease.

[Current advances in leprosy research activities].

Due to the advent of multi-drug therapy (MDT) recommended by the WHO, for the treatment of leprosy, presently, leprosy is regarded as a "curable disease". The number of new cases in Japan is relatively very low, due to which the disease is likely to be neglected, but on scientific grounds, there is a necessity to perform in depth studies. Leprosy caused by M. leprae is still unclear on various aspects including transmission, immunology, nerve damage etc. Here we introduce the recent advances in the field of basic leprosy research.

High frequency of polymorphism Arg753Gln of the Toll-like receptor-2 gene detected by a novel allele-specific PCR.

The recently described family of Toll-like receptors (TLRs) plays a major role in innate immunity by mediating inflammatory reactions against a wide array of pathogens. TLR-2 is reported to interact with various bacterial partial structures including lipoproteins, peptidoglycan, and lipoteichoic acid. Two polymorphisms of the TLR-2 gene have recently been described: Arg753Gln, correlated with the incidence of sepsis in a white population, and Arg677Trp, correlated with the incidence of lepromatous leprosy in an Asian population. Both polymorphisms, when inserted into expression vectors encoding for human TLR-2, reduced stimulation of Chinese hamster ovary cells by synthetic lipopeptides. We furthermore developed a rapid and inexpensive method for the detection of both single nucleotide polymorphisms based on restriction fragment length polymorphism. While no individuals carrying the Arg677Trp SNP were identified in a large group of whites, 9.4% of the study population were found to be heterozygous for the Arg753Gln polymorphism. This ratio is significantly higher than previously reported, and therefore detection of this polymorphism among patients may yield important information for the assessment of risk profiles regarding susceptibility to bacterial infections.

Activation and regulation of Toll-like receptors 2 and 1 in human leprosy.

The expression and activation of Toll-like receptors (TLRs) was investigated in leprosy, a spectral disease in which clinical manifestations correlate with the type of immune response mounted toward Mycobacterium leprae. TLR2-TLR1 heterodimers mediated cell activation by killed M. leprae, indicating the presence of triacylated lipoproteins. A genome-wide scan of M. leprae detected 31 putative lipoproteins. Synthetic lipopeptides representing the 19-kD and 33-kD lipoproteins activated both monocytes and dendritic cells. Activation was enhanced by type-1 cytokines and inhibited by type-2 cytokines. In addition, interferon (IFN)-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced TLR1 expression in monocytes and dendritic cells, respectively, whereas IL-4 downregulated TLR2 expression. TLR2 and TLR1 were more strongly expressed in lesions from the localized tuberculoid form (T-lep) as compared with the disseminated lepromatous form (L-lep) of the disease. These data provide evidence that regulated expression and activation of TLRs at the site of disease contribute to the host defense against microbial pathogens.

Cutting edge: a Toll-like receptor 2 polymorphism that is associated with lepromatous leprosy is unable to mediate mycobacterial signaling.

Toll-like receptors (TLRs) are key mediators of the innate immune response to microbial pathogens. We investigated the role of TLRs in the recognition of Mycobacterium leprae and the significance of TLR2Arg(677)Trp, a recently discovered human polymorphism that is associated with lepromatous leprosy. In mice, TNF-alpha production in response to M. leprae was essentially absent in TLR2-deficient macrophages. Similarly, human TLR2 mediated M. leprae-dependent activation of NF-kappaB in transfected Chinese hamster ovary and human embryonic kidney 293 cells, with enhancement of this signaling in the presence of CD14. In contrast, activation of NF-kappaB by human TLR2Arg(677)Trp was abolished in response to M. leprae and Mycobacterium tuberculosis. The impaired function of this TLR2 variant provides a molecular mechanism for the poor cellular immune response associated with lepromatous leprosy and may have important implications for understanding the pathogenesis of other mycobacterial infections.

Expression of toll-like receptor 2 on human Schwann cells: a mechanism of nerve damage in leprosy

Nerve damage is a clinical hallmark of leprosy and a major source of patient morbidity. We investigated the possibility that human Schwann cells are susceptible to cell death through the activation of Toll-like receptor 2 (TLR2), a pattern recognition receptor of the innate immune system. TLR2 was detected on the surface of human Schwann cell line ST88-14 and on cultured primary human Schwann cells. Activation of the human Schwann cell line and primary human Schwann cell cultures with a TLR2 agonist, a synthetic lipopeptide comprising the N-terminal portion of the putative Mycobacterium leprae 19-kDa lipoprotein, triggered an increase in the number of apoptotic cells. The lipopeptide-induced apoptosis of Schwann cells could be blocked by an anti-TLR2 monoclonal antibody. Schwann cells in skin lesions from leprosy patients were found to express TLR2. It was possible to identify in the lesions Schwann cells that had undergone apoptosis in vivo. The ability of M. leprae ligands to induce the apoptosis of Schwann cells through TLR2 provides a mechanism by which activation of the innate immune response contributes to nerve injury in leprosy.

Expression of Toll-like receptor 2 on human Schwann cells: a mechanism of nerve damage in leprosy.

Nerve damage is a clinical hallmark of leprosy and a major source of patient morbidity. We investigated the possibility that human Schwann cells are susceptible to cell death through the activation of Toll-like receptor 2 (TLR2), a pattern recognition receptor of the innate immune system. TLR2 was detected on the surface of human Schwann cell line ST88-14 and on cultured primary human Schwann cells. Activation of the human Schwann cell line and primary human Schwann cell cultures with a TLR2 agonist, a synthetic lipopeptide comprising the N-terminal portion of the putative Mycobacterium leprae 19-kDa lipoprotein, triggered an increase in the number of apoptotic cells. The lipopeptide-induced apoptosis of Schwann cells could be blocked by an anti-TLR2 monoclonal antibody. Schwann cells in skin lesions from leprosy patients were found to express TLR2. It was possible to identify in the lesions Schwann cells that had undergone apoptosis in vivo. The ability of M. leprae ligands to induce the apoptosis of Schwann cells through TLR2 provides a mechanism by which activation of the innate immune response contributes to nerve injury in leprosy.

Cutting edge: a toll-like receptor 2 polymorphism that is associated with lepromatous leprosy is unable to mediate mycobacterial signaling

Toll-like receptors (TLRs) are key mediators of the innate immune response to microbial pathogens. We investigated the role of TLRs in the recognition of Mycobacterium leprae and the significance of TLR2Arg(677)Trp, a recently discovered human polymorphism that is associated with lepromatous leprosy. In mice, TNF-alpha production in response to M. leprae was essentially absent in TLR2-deficient macrophages. Similarly, human TLR2 mediated M. leprae-dependent activation of NF-kappaB in transfected Chinese hamster ovary and human embryonic kidney 293 cells, with enhancement of this signaling in the presence of CD14. In contrast, activation of NF-kappaB by human TLR2Arg(677)Trp was abolished in response to M. leprae and Mycobacterium tuberculosis. The impaired function of this TLR2 variant provides a molecular mechanism for the poor cellular immune response associated with lepromatous leprosy and may have important implications for understanding the pathogenesis of other mycobacterial infections.

Activation and regulation of toll-like receptors 2 and 1 in human leprosy

The expression and activation of Toll-like receptors (TLRs) was investigated in leprosy, a spectral disease in which clinical manifestations correlate with the type of immune response mounted toward Mycobacterium leprae. TLR2-TLR1 heterodimers mediated cell activation by killed M. leprae, indicating the presence of triacylated lipoproteins. A genome-wide scan of M. leprae detected 31 putative lipoproteins. Synthetic lipopeptides representing the 19-kD and 33-kD lipoproteins activated both monocytes and dendritic cells. Activation was enhanced by type-1 cytokines and inhibited by type-2 cytokines. In addition, interferon (IFN)-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced TLR1 expression in monocytes and dendritic cells, respectively, whereas IL-4 downregulated TLR2 expression. TLR2 and TLR1 were more strongly expressed in lesions from the localized tuberculoid form (T-lep) as compared with the disseminated lepromatous form (L-lep) of the disease. These data provide evidence that regulated expression and activation of TLRs at the site of disease contribute to the host defense against microbial pathogens.

The mannose receptor delivers lipoglycan antigens to endosomes for presentation to T cells by CD1b molecules.

We have characterized the CD1b-mediated presentation pathway for the mycobacterial lipoglycan lipoarabinomannan (LAM) in monocyte-derived antigen-presenting cells. The macrophage mannose receptor (MR) was responsible for uptake of LAM. Antagonism of MR function inhibited both the internalization of LAM and the presentation of this antigen to LAM-reactive T cells. Intracellular MRs were most abundant in early endosomes, but they also were located in the compartment for MHC class II antigen loading (MIIC). Internalized LAM was transported to late endosomes, lysosomes, and MIICs. MRs colocalized with CD1b molecules, suggesting that the MR could deliver LAM to late endosomes for loading onto CD1b. LAM and CD1b colocalized in organelles that may be sites of lipoglycan antigen loading. This pathway links recognition of microbial antigens by a receptor of the innate immune system to the induction of adaptive T cell responses.