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Susceptibility to leprosy is associated with PARK2 and PACRG

Leprosy is caused by Mycobacterium leprae and affects about 700,000 individuals each year. It has long been thought that leprosy has a strong genetic component, and recently we mapped a leprosy susceptibility locus to chromosome 6 region q25–q26 (ref. 3). Here we investigate this region further by using a systematic association scan of the chromosomal interval most likely to harbour this leprosy susceptibility locus. In 197 Vietnamese families we found a significant association between leprosy and 17 markers located in a block of approx. 80 kilobases overlapping the 5′ regulatory region shared by the Parkinsons disease gene PARK2 and the co-regulated gene PACRG. Possession of as few as two of the 17 risk alleles was highly predictive of leprosy. This was confirmed in a sample of 975 unrelated leprosy cases and controls from Brazil in whom the same alleles were strongly associated with leprosy. Variants in the regulatory region shared by PARK2 and PACRG therefore act as common risk factors for leprosy.

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)-γ 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.

Type I Interferon Suppresses Type II Interferon–Triggered Human Anti-Mycobacterial Responses

Interfering with Interferons Infections with Mycobacteria, including Mycobacterium leprae or M. tuberculosis, vary substantially in their clinical presentation. For instance, in some cases of M. leprae, the infection is self-healing with very few lesions. In contrast, some people experience the disseminated form, where skin lesions abound and bacteria are abundant. In patients infected with M. leprae, Teles et al. (p. 1448, published online 28 February) found that the disseminated disease associates with a type I interferon gene signature, whereas the self-healing form associates with a type II interferon gene signature. In cultured cells, type I interferon and its downstream signaling cascade inhibited the antimicrobial response induced by type II interferons, providing a potential explanation for why robust disease rather than protection is seen in some cases of infection. Disseminated Mycobacterium leprae infection is associated with blockade of the antimicrobial response by type I interferons. Type I interferons (IFN-α and IFN-β) are important for protection against many viral infections, whereas type II interferon (IFN-γ) is essential for host defense against some bacterial and parasitic pathogens. Study of IFN responses in human leprosy revealed an inverse correlation between IFN-β and IFN-γ gene expression programs. IFN-γ and its downstream vitamin D–dependent antimicrobial genes were preferentially expressed in self-healing tuberculoid lesions and mediated antimicrobial activity against the pathogen Mycobacterium leprae in vitro. In contrast, IFN-β and its downstream genes, including interleukin-10 (IL-10), were induced in monocytes by M. leprae in vitro and preferentially expressed in disseminated and progressive lepromatous lesions. The IFN-γ–induced macrophage vitamin D–dependent antimicrobial peptide response was inhibited by IFN-β and by IL-10, suggesting that the differential production of IFNs contributes to protection versus pathogenesis in some human bacterial infections.

T-cell release of granulysin contributes to host defense in leprosy

A novel mechanism by which T cells contribute to host defense against microbial pathogens is release of the antimicrobial protein granulysin. We investigated the role of granulysin in human infectious disease using leprosy as a model. Granulysin-expressing T cells were detected in cutaneous leprosy lesions at a six-fold greater frequency in patients with the localized tuberculoid as compared with the disseminated lepromatous form of the disease. In contrast, perforin, a cytolytic molecule that colocalizes with granulysin in cytotoxic granules, was expressed at similar levels across the spectrum of disease. Within leprosy lesions, granulysin colocalized in CD4+ T cells and was expressed in CD4+ T-cell lines derived from skin lesions. These CD4+ T-cell lines lysed targets by the granule exocytosis pathway and reduced the viability of mycobacteria in infected targets. Given the broad antimicrobial spectrum of granulysin, these data provide evidence that T-cell release of granulysin contributes to host defense in human infectious disease.

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

ABSTRACT 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.

Role of Tumor Necrosis Factor–α and Interleukin-10 Promoter Gene Polymorphisms in Leprosy

Single-nucleotide polymorphismswithin thegenescodingfortumornecrosisfactor(TNF)‐a and interleukin (IL)‐10 have been associated with several infectious diseases. To determine whether such polymorphisms are associated with leprosy, genotyping was performed at the 308 and 238 positions of the promoter of the TNF-a gene in 210 and 191 patients with multibacillary (MB) leprosy, respectively; 90 and 79 patients with paucibacillary (PB) leprosy; and 92 control subjects. For the 592 and 819 positions within the promoter of the IL-10 gene, 143 patients with MB leprosy, 79 patients with PB leprosy, and 62 control subjects were included in the analysis. TNF2 allele frequency was significantly higheramong controlsubjects than among all patients with leprosy or in the MB group ( and ). For the ILP ! .05 P ! .01 10 gene, the frequency of the homozygous 819TT genotype was significantly higher among patients than among control subjects. These data indicate that a relationship exists between TNF-a and IL-10 promoter polymorphisms and the development of PB leprosy. The more benign paucibacillary (PB) forms of leprosy— borderline tuberculoid (BT) and tuberculoid tuberculoid (TT) leprosy—are characterized by the predominance of a Th1-type immune response, the presence of well-formed granulomas at the site of the lesion, and control of mycobacterial replication. In contrast, in the multibacillary (MB) forms—borderline borderline (BB) and borderline lepromatous (BL) leprosy and lepromatous leprosy (LL)—no granuloma is seen, and high bacterial load and antibody levels are detected. Cytokines evidently play a critical role in triggering host-pathogen interactions. On one hand, greater tumor necrosis factor (TNF)–a production,

MicroRNA-21 targets the vitamin D-dependent antimicrobial pathway in leprosy.

Leprosy provides a model to investigate mechanisms of immune regulation in humans, given that the disease forms a spectrum of clinical presentations that correlate with host immune responses. Here we identified 13 miRNAs that were differentially expressed in the lesions of subjects with progressive lepromatous (L-lep) versus the self-limited tuberculoid (T-lep) disease. Bioinformatic analysis revealed a significant enrichment of L-lep–specific miRNAs that preferentially target key immune genes downregulated in L-lep versus T-lep lesions. The most differentially expressed miRNA in L-lep lesions, hsa-mir-21, was upregulated in Mycobacterium leprae–infected monocytes. By directly downregulating Toll-like receptor 2/1 heterodimer (TLR2/1)-induced CYP27B1 and IL1B expression as well as indirectly upregulating interleukin-10 (IL-10), hsa-mir-21 inhibited expression of the genes encoding two vitamin D–dependent antimicrobial peptides, CAMP and DEFB4A. Conversely, knockdown of hsa-mir-21 in M. leprae–infected monocytes enhanced expression of CAMP and DEFB4A and restored TLR2/1-mediated antimicrobial activity against M. leprae. Therefore, the ability of M. leprae to upregulate hsa-mir-21 targets multiple genes associated with the immunologically localized disease form, providing an effective mechanism to escape from the vitamin D–dependent antimicrobial pathway.

Divergence of macrophage phagocytic and antimicrobial programs in leprosy

Effective innate immunity against many microbial pathogens requires macrophage programs that upregulate phagocytosis and direct antimicrobial pathways, two functions generally assumed to be coordinately regulated. We investigated the regulation of these key functions in human blood-derived macrophages. Interleukin-10 (IL-10) induced the phagocytic pathway, including the C-type lectin CD209 and scavenger receptors, resulting in phagocytosis of mycobacteria and oxidized low-density lipoprotein. IL-15 induced the vitamin D-dependent antimicrobial pathway and CD209, yet the cells were less phagocytic. The differential regulation of macrophage functional programs was confirmed by analysis of leprosy lesions: the macrophage phagocytosis pathway was prominent in the clinically progressive, multibacillary form of the disease, whereas the vitamin D-dependent antimicrobial pathway predominated in the self-limited form and in patients undergoing reversal reactions from the multibacillary to the self-limited form. These data indicate that macrophage programs for phagocytosis and antimicrobial responses are distinct and differentially regulated in innate immunity to bacterial infections.

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.