Daniela R. Faria

Decreased In Situ Expression of Interleukin-10 Receptor Is Correlated with the Exacerbated Inflammatory and Cytotoxic Responses Observed in Mucosal Leishmaniasis

ABSTRACT Human infection with Leishmania braziliensis can lead to cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). We hypothesize that the intense tissue destruction observed in ML is a consequence of an uncontrolled exacerbated inflammatory immune response, with cytotoxic activity. For the first time, this work identifies the cellular sources of inflammatory and antiinflammatory cytokines, the expression of effector molecules, and the expression of interleukin-10 (IL-10) receptor in ML and CL lesions by using confocal microscopy. ML lesions displayed a higher number of gamma interferon (IFN-γ)-producing cells than did CL lesions. In both ML and CL, CD4+ cells represented the majority of IFN-γ-producing cells, followed by CD8+ cells and CD4− CD8− cells. The numbers of tumor necrosis factor alpha-positive cells, as well as those of IL-10-producing cells, were similar in ML and CL lesions. The effector molecule granzyme A showed greater expression in ML than in CL lesions, while inducible nitric oxide synthase did not. Finally, the expression of IL-10 receptor was lower in ML than in CL lesions. Thus, our data identified distinct cytokine and cell population profiles for CL versus ML patients and provide a possible mechanism for the development of ML disease through the demonstration that low expression of IL-10 receptor is present in conjunction with a cytotoxic and inflammatory profile in ML.

Recruitment of CD8+ T cells expressing granzyme A is associated with lesion progression in human cutaneous leishmaniasis

Human infection with Leishmania braziliensis leads to the establishment of cutaneous leishmaniasis (CL), characterized by the appearance of skin lesions that progress from nonulcerated to ulcerated forms. Our goal was to characterize the immunological kinetics associated with this progression, comparing the cellular composition, cytokines and granzyme expression between lesions of patients with early (E‐CL) and late stages (L‐CL) of CL. Histopathological analysis showed that lesions from L‐CL had more exuberant inflammatory infiltrate as compared to E‐CL. Although E‐CL and L‐CL lesions were predominantly mononuclear, lesions from E‐CL patients presented higher neutrophil and eosinophil counts than L‐CL. While percentages of CD4+ and of CD68+ cells were slightly higher in L‐CL, a fivefold increase of CD8+ cells was observed in L‐CL, as compared to E‐CL. Moreover, CD8+ T‐cells from L‐CL expressed significantly higher levels of granzyme A than E‐CL. Interestingly, granzyme A expression was positively correlated with intensity of the inflammatory infiltrate in L‐CL but not E‐CL. Lastly, percentages of IFN‐γ+ and IL‐10+ cells were higher in L‐CL as compared to E‐CL, with CD4+ T‐cells and CD68+ monocytes as the main sources of these cytokines, respectively. These results suggest that recruitment of CD8+ granzyme A+ T cells is involved in lesion progression in human CL.

Interleukin 17 Production among Patients with American Cutaneous Leishmaniasis

Interleukin 17 (IL-17) plays a critical role in inflammation and autoimmunity. Very little is known about IL-17 in protozoa infection. Here, we show that lymphocytes obtained from patients with mucosal leishmaniasis and cutaneous leishmaniasis produce higher levels of IL-17 than do lymphocytes obtained from uninfected control subjects (P<.01). There was a tendency for tissue obtained from patients with mucosal leishmaniasis to contain a higher number of cells expressing IL-17, compared with tissue obtained from patients with cutaneous leishmaniasis, and there was a direct correlation between the number of cells expressing IL-17 and the presence of cellular inflammation at the lesion site (r2 = 0.86; P<.001) These data support the role of IL-17 in the pathogenesis of the inflammatory reaction in leishmaniasis.

Cutaneous Leishmaniasis during Pregnancy: Exuberant Lesions and Potential Fetal Complications

Cutaneous leishmaniasis affects millions of people worldwide. After observations of atypical lesions in pregnant women at the health centers of Corte de Pedra, Brazil, 9 years of records were reviewed, and 26 pregnant patients were identified. A retrospective case-control study revealed that lesions in pregnant women were much larger than those in nonpregnant patients in an age- and sex-matched group (mean area, 6.08 cm2 vs. 1.46 cm2; P=.008), and many lesions had an exophytic nature. Despite foregoing treatment until after delivery, response to pentavalent antimony therapy was favorable (rate of cure with 1 course of treatment, 85%). High rates of preterm births (10.5%) and stillbirths (10.5%) were reported. Cutaneous leishmaniasis during pregnancy produces distinct lesions and may have adverse fetal effects.

Immunoregulatory mechanisms and CD4−CD8− (double negative) T cell subpopulations in human cutaneous leishmaniasis: A balancing act between protection and pathology

Cellular immune responses directed against protozoan parasites are key for controlling pathogen replication and disease resolution. However, an uncontrolled, or improperly controlled, response can be deleterious to the host in terms of both allowing for the establishment of pathology, as well as less effective establishment of memory responses. Human cutaneous leishmaniasis is a disease caused by the infection with Leishmania spp. following a bite from the sandfly, the natural vector of this disease. Tens of millions worldwide are currently infected with Leishmania and no effective vaccines have been developed to date. In the face of the complexity presented by the interaction between a host (humans) with the parasite, Leishmania, and the fact that this parasite is inoculated by another complex, biologically active, vector, the sandfly, it is clearly important to study the immunoregulatory mechanisms that are induced in humans naturally infected by this parasite if we hope to develop effective vaccines and immunotherapeutic treatments in the future. Our laboratory has focused over the years on the study of the local and systemic T cell response during the first episode of cutaneous leishmaniasis suffered by individuals before they undergo antimony treatment. The goal of this review is to briefly outline our findings with hopes of putting our most recent studies concerning the dichotomy between alpha/beta TCR and gamma/delta TCR expressing, CD4-CD8- (double negative-DN) T cells in the context of a balanced immune response against Leishmania and to discuss the implications of these findings toward our understanding of human leishmaniasis.

A signal peptide mutation of the arginine vasopressin gene in monozygotic twins

Familial neurohypophysial diabetes insipidus (FNDI) is a rare autosomal dominant syndrome stemming from the absence of arginine vasopressin (AVP). More than thirty‐five different germline mutations in the arginine vasopressin‐neurophysin II gene have been reported. These mutations are either in the signal peptide or scattered throughout the neurophysin II domain. A missense mutation altering alanine at position −1 to either valine or threonine in the signal peptide domain has previously been found in ten unrelated families. In the present report, Brazilian female monozygotic twins with clinically typical central DI in whom biochemical and molecular characterization were carried out are described. Direct mutational analysis by sequencing of the vasopressin gene in germline DNA revealed a heterozygous missense mutation (G→A) at nucleotide 279, predicting the substitution of alanine by threonine at position −1 of the signal peptide moiety. In summary, we present an extremely rare case of familial central diabetes insipidus in monozygotic Brazilian twins with a seemingly common missense mutation in the AVP gene.

Histopathological outcome of Leishmania major-infected BALB/c mice is improved by oral treatment with N-acetyl-l-cysteine.

Leishmania major infected BALB/c mice were treated with N‐acetyl‐l‐cysteine (NAC), a glutathione precursor, to evaluate the role of in vivo glutathione on lesion pathology and cytokine profiles following infection. Mice were maintained on NAC‐containing water 2 days before infection for a total of 14 weeks. The BALB/c response to L. major infection was improved by oral administration of NAC, at the level of histopathological outcome, lesion progression and cytokine profile. A significantly improved histopathological outcome of the footpad lesion, characterized by a mixed inflammatory infiltrate organized in a focal pattern with little tissue destruction and a reduced parasite load, was observed in NAC‐treated BALB/c mice. Histopathological modulation was accompanied by a modified cytokine pattern from popliteal lymph node cells, demonstrated by a sustained higher frequency of interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α)‐producing cells. This work points to an important role for glutathione in the modulation of effector responses in BALB/c mice.

CD4+ T cells defined by their Vβ T cell receptor expression are associated with immunoregulatory profiles and lesion size in human leishmaniasis

Leishmaniasis is caused by infection with the protozoan parasite, Leishmania, that parasitizes human cells, and the cellular immune response is essential for controlling infection. In order to measure the host T cell response to Leishmania infection, we have measured the expansion, activation state and functional potential of specific T cells as identified by their T cell receptor Vβ region expression. In a group of cutaneous leishmaniasis (CL) patients, we evaluated these characteristics in nine different T cell subpopulations as identified by their Vβ region expression, before and after specific Leishmania antigen stimulation. Our results show: (1) an increase in CD4+ T cells expressing Vβ 5·2 and Vβ 24 in CL compared to controls; (2) a Leishmania antigen‐induced increase in CD4+ T cells expressing Vβ 5·2, 11, 12 and 17; (3) a profile of previous activation of CD4+ Vβ 5·2‐, 11‐ and 24‐positive T cells, with higher expression of CD45RO, HLA‐DR, interferon‐γ, tumour necrosis factor‐α and interleukin‐10 compared to other Vβ‐expressing subpopulations; (4) a positive correlation between higher frequencies of CD4+Vβ5·2+ T cells and larger lesions; and (5) biased homing of CD4+ T cells expressing Vβ 5·2 to the lesion site. Given that CL disease involves a level of pathology (ulcerated lesions) and is often followed by long‐lived protection and cure, the identification of specific subpopulations active in this form of disease could allow for the discovery of immunodominant Leishmania antigens important for triggering efficient host responses against the parasite, or identify cell populations most involved in pathology.

Immunoregulatory and Effector Activities in Human Cutaneous and Mucosal Leishmaniasis: Understanding Mechanisms of Pathology

Individuals infected with Leishmania braziliensis may develop the relatively benign localized cutaneous (CL) form or the mucosal (ML) form of the disease, which represents a more severe and mutilating variation. Interaction between parasite and host cells, as well as the genetic background of the host, are important determinants of the immune response, which is critical in determining disease outcome. Our studies over the years have been designed to determine the immunoregulatory and effector functions that culminate in the formation of lesions in CL and ML disease and how these host response factors may be better understood for design of novel therapies and prophylaxis. By studying the immune response from CL and ML patients in both the peripheral blood and in situ, we have learned much concerning the dynamics of the host–pathogen interaction that leads to the development of CL and ML. We used multiparameter flow cytometry to study the immunoregulatory profiles of the peripheral blood leukocytes, as well as laser scanning confocal microscopy to examine in situ several aspects of the local response, including the intensity of the inflammatory infiltrate, the cellular composition, inflammatory and anti‐inflammatory cytokine expression, and the expression of the effector cytotoxic molecule, granzyme A, in lesions from CL and ML patients. Moreover, the application of correlative analysis between these immunological parameters has helped shed light on disease progression in CL and ML. These findings are reviewed within the context of understanding cellular and molecular mechanisms associated with the development of pathology in these diseases through a comparative analysis of the clinical forms, CL and ML, as well as of studies derived from peripheral blood and lesions. Drug Dev Res 72:430–436, 2011.

Distinct Roles of Th17 and Th1 Cells in Inflammatory Responses Associated with the Presentation of Paucibacillary Leprosy and Leprosy Reactions Th17 Immune Response in Leprosy

It is well established that helper T cell responses influence resistance or susceptibility to Mycobacterium leprae infection, but the role of more recently described helper T cell subsets in determining severity is less clear. To investigate the involvement of Th17 cells in the pathogenesis of leprosy, we determined the immune profile with variant presentations of leprosy. Firstly, IL‐17A, IFN‐γ and IL‐10 were evaluated in conjunction with CD4+ T cell staining by confocal microscopy of lesion biopsies from tuberculoid (TT) and lepromatous leprosy (LL) patients. Secondly, inflammatory cytokines were measured by multiplex assay of serum samples from Multibacillary (MB, n = 28) and Paucibacillary (PB, n = 23) patients and household contacts (HHC, n = 23). Patients with leprosy were also evaluated for leprosy reaction occurrence: LR+ (n = 8) and LR‐ (n = 20). Finally, peripheral blood mononuclear cells were analysed by flow cytometry used to determine the phenotype of cytokine‐producing cells. Lesions from TT patients were found to have more CD4+IL‐17A+ cells than those from LL patients. Higher concentrations of IL‐17A and IL‐1β were observed in serum from PB than MB patients. The highest serum IFN‐γ concentrations were, however, detected in sera from MB patients that developed leprosy reactions (MB LR+). Together, these results indicate that Th1 cells were associated with both the PB presentation and also with leprosy reactions. In contrast, Th17 cells were associated with an effective inflammatory response that is present in the PB forms but were not predictive of leprosy reactions in MB patients.