Roberta Olmo Pinheiro

Capsular polysaccharides galactoxylomannan and glucuronoxylomannan from Cryptococcus neoformans induce macrophage apoptosis mediated by Fas ligand.

The effects of capsular polysaccharides, galactoxylomannan (GalXM) and glucuronoxylomannan (GXM), from acapsular (GXM negative) and encapsulate strains of Cryptococcus neoformans were investigated in RAW 264.7 and peritoneal macrophages. Here, we demonstrate that GalXM and GXM induced different cytokines profiles in RAW 264.7 macrophages. GalXM induced production of TNF‐α, NO and iNOS expression, while GXM predominantly induced TGF‐β secretion. Both GalXM and GXM induced early morphological changes identified as autophagy and late macrophages apoptosis mediated by Fas/FasL interaction, a previously unidentified mechanism of virulence. GalXM was more potent than GXM at induction of Fas/FasL expression and apoptosis on macrophages in vitro and in vivo. These findings uncover a mechanism by which capsular polysaccharides from C. neoformans might compromise host immune responses.

Induction of autophagy correlates with increased parasite load of Leishmania amazonensis in BALB/c but not C57BL/6 macrophages.

We investigated the role of autophagy in infection of macrophages by Leishmania amazonensis. Induction of autophagy by IFN-gamma or starvation increased intracellular parasite load and the percentages of infected macrophages from BALB/c but not from C57BL/6 mice. In contrast, starvation did not affect the replication of either Leishmania major or Trypanosoma cruzi in BALB/c macrophages. In BALB/c macrophages, starvation resulted in increased monodansylcadaverine staining and in the appearance of double-membrane and myelin-like vesicles characteristic of autophagosomes. Increased parasite load was associated with a reduction in NO levels and was attenuated by wortmannin, an inhibitor of autophagy. In infected macrophages from BALB/c, but not from C57BL/6 mice, starvation increased the number of lipid bodies and the amounts of PGE(2) produced. Exogenous PGE(2) increased parasite load in macrophages from BALB/c, but not C57BL/6 mice. The cyclooxygenase inhibitor indomethacin prevented the increase of parasite load in starved BALB/c macrophages, and actually induced parasite killing. These results suggest that autophagy regulates the outcome of L. amazonensis infection in macrophages in a host strain specific manner.

TLR6-Driven Lipid Droplets in Mycobacterium leprae-Infected Schwann Cells: Immunoinflammatory Platforms Associated with Bacterial Persistence

The mechanisms responsible for nerve injury in leprosy need further elucidation. We recently demonstrated that the foamy phenotype of Mycobacterium leprae-infected Schwann cells (SCs) observed in nerves of multibacillary patients results from the capacity of M. leprae to induce and recruit lipid droplets (LDs; also known as lipid bodies) to bacterial-containing phagosomes. In this study, we analyzed the parameters that govern LD biogenesis by M. leprae in SCs and how this contributes to the innate immune response elicited by M. leprae. Our observations indicated that LD formation requires the uptake of live bacteria and depends on host cell cytoskeleton rearrangement and vesicular trafficking. TLR6 deletion, but not TLR2, completely abolished the induction of LDs by M. leprae, as well as inhibited the bacterial uptake in SCs. M. leprae-induced LD biogenesis correlated with increased PGE2 and IL-10 secretion, as well as reduced IL-12 and NO production in M. leprae-infected SCs. Analysis of nerves from lepromatous leprosy patients showed colocalization of M. leprae, LDs, and cyclooxygenase-2 in SCs, indicating that LDs are sites for PGE2 synthesis in vivo. LD biogenesis Inhibition by the fatty acid synthase inhibitor C-75 abolished the effect of M. leprae on SC production of immunoinflammatory mediators and enhanced the mycobacterial-killing ability of SCs. Altogether, our data indicated a critical role for TLR6-dependent signaling in M. leprae–SC interactions, favoring phagocytosis and subsequent signaling for induction of LD biogenesis in infected cells. Moreover, our observations reinforced the role of LDs favoring mycobacterial survival and persistence in the nerve. These findings give further support to a critical role for LDs in M. leprae pathogenesis in the nerve.

Intranasal Vaccination against Cutaneous Leishmaniasis with a Particulated Leishmanial Antigen or DNA Encoding LACK

ABSTRACT We have previously demonstrated that oral delivery of a disease-promoting particulated antigen of Leishmania amazonensis (LaAg) partially protects mice against cutaneous leishmaniasis. In the present work, we sought to optimize a mucosal vaccine by using the intranasal route for delivery of different antigen preparations, including (i) LaAg, (ii) soluble recombinant p36/LACK leishmanial antigen (LACK), and (iii) plasmid DNA encoding LACK (LACK DNA). BALB/c mice that received two intranasal doses of 10 μg of LaAg and were challenged 1 week postvaccination with L. amazonensis developed delayed but effective control of lesion growth. A diminished parasite burden was accompanied by enhancement of both gamma interferon (IFN-γ) and interleukin-10 levels in the lesion-draining lymph nodes. The vaccine efficacy improved with time. At 4 months postvaccination, when a strong parasite-specific TH1-type response was present in vivo, the infection was controlled for at least 5 months after challenge. In contrast to the particulated LaAg, soluble LACK (10 μg/dose) had no effect. Interestingly, LACK DNA (30 μg/dose), but not empty DNA, promoted rapid and durable protective immunity. Parasite growth was effectively controlled, and at 5 months after challenge LACK-reactive cells in both the mucosal and lesion-draining lymph nodes produced high levels of IFN-γ. These results demonstrate for the first time the feasibility of using the intranasal route for long-lived memory vaccination against cutaneous leishmaniasis with adjuvant-free crude antigens or DNA.

Mycobacterium leprae–host-cell interactions and genetic determinants in leprosy: an overview

Leprosy, also known as Hansens disease, is a chronic infectious disease caused by Mycobacterium leprae in which susceptibility to the mycobacteria and its clinical manifestations are attributed to the host immune response. Even though leprosy prevalence has decreased dramatically, the high number of new cases indicates active transmission. Owing to its singular features, M. leprae infection is an attractive model for investigating the regulation of human immune responses to pathogen-induced disease. Leprosy is one of the most common causes of nontraumatic peripheral neuropathy worldwide. The proportion of patients with disabilities is affected by the type of leprosy and delay in diagnosis. This article briefly reviews the clinical features as well as the immunopathological mechanisms related to the establishment of the different polar forms of leprosy, the mechanisms related to M. leprae-host cell interactions and prophylaxis and diagnosis of this complex disease. Host genetic factors are summarized and the impact of the development of interventions that prevent, reverse or limit leprosy-related nerve impairments are discussed.

CD163 favors Mycobacterium leprae survival and persistence by promoting anti-inflammatory pathways in lepromatous macrophages

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 Myco‐bacterium 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 myco‐bacterial entry and survival.

The role of indoleamine 2, 3-dioxygenase in lepromatous leprosy immunosuppression

To elucidate further the possible role of the tryptophan, rate‐limiting enzyme indoleamine 2, 3‐dioxygenase (IDO) in leprosy, the distribution of IDO‐positive cells and IDO activity in the skin biopsies and sera of these patients representing the entire spectrum of the disease were studied. An increased number of macrophages/dendritic cells (DC–lineage IDO+ cells were found in lepromatous (LL) compared to tuberculoid (BT) and reversal reaction (RR) patients. IDO‐positive cells showing CD68 and CD86 surface markers predominated in LL lesions, while higher levels of IDO activity were observed in the sera of LL versus BT patients. Tests revealed an increased IDO message in Mycobacterium leprae‐stimulated peripheral blood mononuclear cells (PBMC) by real‐time polymerase chain reaction (PCR) and increased IDO expression in M. leprae‐stimulated CD14+ cells of both healthy controls (HC) and LL patients, as evaluated via flow cytometry. Increased M. leprae‐induced IDO–protein synthesis was also confirmed by Western blot. Based on our in vitro studies, it was confirmed that M. leprae up‐regulated IDO expression and activity in HC and LL monocytes. Interferon (IFN)‐γ synergized with M. leprae in promoting IDO expression and activity in monocytes. IDO expression induced by both IFN‐γ and M. leprae was abrogated by 1‐methyltryptophan (1‐MT). Our data suggest that M. leprae chronic infection activates the suppressive molecule IDO which, in turn, contributes to the specific immunosuppression observed in LL leprosy.

Effect of Apoptotic Cell Recognition on Macrophage Polarization and Mycobacterial Persistence

ABSTRACT Intracellular Mycobacterium leprae infection modifies host macrophage programming, creating a protective niche for bacterial survival. The milieu regulating cellular apoptosis in the tissue plays an important role in defining susceptible and/or resistant phenotypes. A higher density of apoptotic cells has been demonstrated in paucibacillary leprosy lesions than in multibacillary ones. However, the effect of apoptotic cell removal on M. leprae-stimulated cells has yet to be fully elucidated. In this study, we investigated whether apoptotic cell removal (efferocytosis) induces different phenotypes in proinflammatory (Mϕ1) and anti-inflammatory (Mϕ2) macrophages in the presence of M. leprae. We stimulated Mϕ1 and Mϕ2 cells with M. leprae in the presence or absence of apoptotic cells and subsequently evaluated the M. leprae uptake, cell phenotype, and cytokine pattern in the supernatants. In the presence of M. leprae and apoptotic cells, Mϕ1 macrophages changed their phenotype to resemble the Mϕ2 phenotype, displaying increased CD163 and SRA-I expression as well as higher phagocytic capacity. Efferocytosis increased M. leprae survival in Mϕ1 cells, accompanied by reduced interleukin-15 (IL-15) and IL-6 levels and increased transforming growth factor beta (TGF-β) and IL-10 secretion. Mϕ1 cells primed with M. leprae in the presence of apoptotic cells induced the secretion of Th2 cytokines IL-4 and IL-13 in autologous T cells compared with cultures stimulated with M. leprae or apoptotic cells alone. Efferocytosis did not alter the Mϕ2 cell phenotype or cytokine secretion profile, except for TGF-β. Based on these data, we suggest that, in paucibacillary leprosy patients, efferocytosis contributes to mycobacterial persistence by increasing the Mϕ2 population and sustaining the infection.

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.

Type 1 reaction in leprosy: a model for a better understanding of tissue immunity under an immunopathological condition.

Type 1 reaction (T1R) or reversal reaction is the leading cause of physical disabilities and deformities in leprosy. Leprosy patients, even after being considered cured and released from treatment, may suffer from reactional episodes for long periods of time. Early diagnosis is a great challenge for effectively treating and managing T1R. There is an urgent need to identify the most significant biomarkers to prevent recurrent T1R and to differentiate late T1R from relapse. T1R continues to be treated with corticosteroids and complications due to iatrogenic treatment remain frequent. This review aims to provide a framework from which to approach the great challenges that still persist in T1R management and debate key issues in order to reduce the distance between basic research and the clinic.