Lepromatous macrophages possess a regulatory phenotype that contributes to the immunosuppression observed in leprosy. CD163, a scavenger receptor that recognizes hemoglobin-haptoglobin complexes, is expressed at higher levels in lepromatous cells, although its functional role in leprosy is not yet established. We herein demonstrate that human lepromatous lesions are microenvironments rich in IDO + CD163 + . Cells isolated from these lesions were CD68 + IDO + CD163 + while higher levels of sCD163 in lepromatous sera positively correlated with IL-10 levels and IDO activity. Different Mycobacterium leprae (ML) concentrations in healthy monocytes likewise revealed a positive correlation between increased concentrations of the mycobacteria and IDO, CD209, and CD163 expression. The regulatory phenotype in ML-stimulated monocytes was accompanied by increased TNF, IL-10, and TGF-β levels whereas IL-10 blockade reduced ML-induced CD163 expression. The CD163 blockade reduced ML uptake in human monocytes. ML uptake was higher in HEK293 cells transfected with the cDNA for CD163 than in untransfected cells. Simultaneously, increased CD163 expression in lepromatous cells seemed to be dependent on ML uptake, and contributed to augmented iron storage in lepromatous macrophages. Altogether, these results suggest that ML-induced CD163 expression modulates the host cell phenotype to create a favorable environment for mycobacterial entry and survival.Keywords: CD163 r IL-10 r Leprosy r Macrophages IntroductionLeprosy is an infectious disease caused by Mycobacterium leprae (ML) in which susceptibility to the mycobacteria and its clinicalmanifestations are attributed to the host immune response. The clinical and immunological patterns of this unique chronic infectious disease clearly demonstrate a continuous scale of changes in histological lesions. Disease classification is defined Correspondence: Dr. Euzenir N. Sarno e-mail: euzenir@fiocruz.br within two poles (tuberculoid to lepromatous) with transitions between these clinical forms. While typical epithelioid macrophages predominate at the paucibacillary tuberculoid pole of the disease, inactivated foamy macrophages predominate at the lepromatous end [1]. In lepromatous leprosy (LL), the lack of systemic inflammatory signals and corresponding local ones strongly indicates that a complex anti-inflammatory network is at work. In this * These authors contributed equally to this work as first authors. * * These authors contributed equally to this work as senior authors. Eur. J. Immunol. 2012. 42: 2925-2936 regard, neuroendocrine system involvement, in conjunction with the existence of multiple suppressive pathways under the control of the innate and adaptive immune response, has been reported [2][3][4][5][6][7].We have suggested that IDO may play a role in a hitherto unknown suppressive mechanism in leprosy [6]. It has also been reported that accumulated oxidized host phospholipids in lepromatous macrophages downregulate the innate immune response [8]. Foamy macrophages seem to sust...

SUMMARYA diverse range of infectious organisms, including mycobacteria, have been reported to induce cell death in vivo and in vitro. Although morphological features of apoptosis have been identi®ed in leprosy lesions, it has not yet been determined whether Mycobacterium leprae modulates programmed cell death. For that purpose, peripheral blood mononuclear cells obtained from leprosy patients were stimulated with different concentrations of this pathogen. Following analysis by¯ow cytometry on 7AAD/CD14 cells, it was observed that M. leprae induced apoptosis of monocyte-derived macrophages in a dose-dependent manner in both leprosy patients and healthy individuals, but still with lower ef®ciency as compared to M. tuberculosis. Expression of tumour necrosis factor-a (TNF-a), Bax-a, Bak mRNA and TNF-a protein was also detected in these cultures; in addition, an enhancement in the rate of apoptotic cells (and of TNF-a release) was noted when interferon-g was added to the wells. On the other hand, incubation of the cells with pentoxifylline impaired mycobacterium-induced cell death, the secretion of TNF-a, and gene expression in vitro. In addition, diminished bacterial entry decreased both TNF-a levels and the death of CD14 cells, albeit to a different extent. When investigating leprosy reactions, an enhanced rate of spontaneous apoptosis was detected as compared to the unreactive lepromatous patients. The results demonstrated that M. leprae can lead to apoptosis of macrophages through a mechanism that could be at least partially related to the expression of pro-apoptotic members of the Bcl-2 protein family and of TNF-a. Moreover, while phagocytosis may be necessary, it seems not to be crucial to the induction of cell death by the mycobacteria.

Using a short-term bulk culture protocol designed for an intracellularstaining method based on a flow cytometry approach to the frequencies of cytokine-producing cells from tuberculosis and leprosy patients, we found distinct patterns of T cell subset expression. The method also reveals the profile of peak cytokine production and can provide simultaneous information about the phenotype of cytokineproducing cells, providing a reliable assay for monitoring the immunity of these patients. The immune response of Mycobacterium leprae and purified protein derivative (PPD) in vitro to a panel of mycobacteria-infected patients from an endemic area was assessed in primary mononuclear cell cultures. The kinetics and source of the cytokine pattern were measured at the single-cell level. IFN-γ-, TNF-α-, IL-4-and IL-10-secreting T cells were intracytoplasmic evaluated in an attempt to identify M. leprae-and PPD-specific cells directly from the peripheral blood. The analysis by this approach indicated that TNF-α was the first (8 h) to be produced, followed by IFN-γ (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-α than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-γ. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations.

The enzyme IDO-1 is involved in the first stage of tryptophan catabolism and has been described in both microbicidal and tolerogenic microenvironments. Previous data from our group have shown that IDO-1 is differentially regulated in the distinctive clinical forms of leprosy. The present study aims to investigate the mechanisms associated with IDO-1 expression and activity in human monocyte-derived dendritic cells (mDCs) after stimulation with irradiated Mycobacterium leprae and its fractions. M. leprae and its fractions induced the expression and activity of IDO-1 in human mDCs. Among the stimuli studied, irradiated M. leprae and its membrane fraction (MLMA) induced the production of proinflammatory cytokines TNF and IL-6 whereas irradiated M. leprae and its cytosol fraction (MLSA) induced an increase in IL-10. We investigated if TLR2 activation was necessary for IDO-1 induction in mDCs. We observed that in cultures treated with a neutralizing anti-TLR2 antibody, there was a decrease in IDO-1 activity and expression induced by M. leprae and MLMA. The same effect was observed when we used a MyD88 inhibitor. Our data demonstrate that coculture of mDCs with autologous lymphocytes induced an increase in regulatory T (Treg) cell frequency in MLSA-stimulated cultures, showing that M. leprae constituents may play opposite roles that may possibly be related to the dubious effect of IDO-1 in the different clinical forms of disease. Our data show that M. leprae and its fractions are able to differentially modulate the activity and functionality of IDO-1 in mDCs by a pathway that involves TLR2, suggesting that this enzyme may play an important role in leprosy immunopathogenesis.