Macrophage immunophenotypes in Jorge Lobo's disease and lepromatous leprosy- A comparative study.

Jorge Lobo's disease (JLD) and lepromatous leprosy (LL) share several clinical, histological and immunological features, especially a deficiency in the cellular immune response. Macrophages participate in innate and adaptive inflammatory immune responses, as well as in tissue regeneration and repair. Macrophage function deficiency results in maintenance of diseases. M1 macrophages produce pro-inflammatory mediators and M2 produce anti-inflammatory cytokines. To better understand JLD and LL pathogenesis, we studied the immunophenotype profile of macrophage subtypes in 52 JLD skin lesions, in comparison with 16 LL samples, using a panmacrophage (CD68) antibody and selective immunohistochemical markers for M1 (iNOS) and M2 (CD163, CD204) responses, HAM56 (resident/fixed macrophage) and MAC 387 (recently infiltrating macrophage) antibodies. We found no differences between the groups regarding the density of the CD163, CD204, MAC387+ immunostained cells, including iNOS, considered a M1 marker. But HAM56+ cell density was higher in LL samples. By comparing the M2 and M1 immunomarkers in each disease separately, some other differences were found. Our results reinforce a higher M2 response in JLD and LL patients, depicting predominant production of anti-inflammatory cytokines, but also some distinction in degree of macrophage activation. Significant amounts of iNOS + macrophages take part in the immune milieu of both LL and JLD samples, displaying impaired microbicidal activity, like alternatively activated M2 cells.

M2-Polarized Macrophages Determine Human Cutaneous Lesions in Lacaziosis.

Lacaziosis is a cutaneous chronic mycosis caused by Lacazia loboi. Macrophages are important cells in the host immune response in fungal infections. The macrophage population exhibits strong plasticity that varies according to the stimuli in the microenvironment of lesions M1 profile promotes a Th1 pattern of cytokines and a microbicidal function and M2 is related to Th2 cytokines and immunomodulatory response. We investigated the population of M1 and M2 polarized macrophages in human cutaneous lesions. A total of 27 biopsies from human lesions were submitted to an immunohistochemistry protocol using antibodies to detect M1 and M2 macrophages (Arginase-1, CD163, iNOS, RBP-J and cMAF). We could observe high number of cells expressing Arginase1, CD163 and c-MAF that correspond to elements of the M2 profile of macrophage, over iNOS and RBP-J (elements of the M1 profile). The results suggest a predominant phenotype of M2 macrophages, which have an immunomodulatory role and probably contributing to chronicity of Lacaziosis.

Comparative Innate and Adaptive Immune Responses in Atlantic Bottlenose Dolphins (Tursiops truncatus) With Viral, Bacterial, and Fungal Infections.

Free-ranging Atlantic bottlenose dolphins (n = 360) from two southeastern U.S. estuarine sites were given comprehensive health examinations between 2003 and 2015 as part of a multi-disciplinary research project focused on individual and population health. The study sites (and sample sizes) included the Indian River Lagoon (IRL), Florida, USA (n = 246) and Charleston harbor and associated rivers (CHS), South Carolina, USA (n = 114). Results of a suite of clinicoimmunopathologic tests revealed that both populations have a high prevalence of infectious and neoplastic disease and a variety of abnormalities of their innate and adaptive immune systems. Subclinical infections with cetacean morbillivirus and Chlamydiaceae were detected serologically. Clinical evidence of orogenital papillomatosis was supported by the detection of a new strain of dolphin papillomavirus and herpesvirus by molecular pathology. Dolphins with cutaneous lobomycosis/lacaziasis were subsequently shown to be infected with a novel, uncultivated strain of Paracoccidioides brasiliensis, now established as the etiologic agent of this enigmatic disease in dolphins. In this review, innate and adaptive immunologic responses are compared between healthy dolphins and those with clinical and/or immunopathologic evidence of infection with these specific viral, bacterial, and fungal pathogens. A wide range of immunologic host responses was associated with each pathogen, reflecting the dynamic and complex interplay between the innate, humoral, and cell-mediated immune systems in the dolphin. Collectively, these studies document the comparative innate and adaptive immune responses to various types of infectious diseases in free-ranging Atlantic bottlenose dolphins. Evaluation of the type, pattern, and degree of immunologic response to these pathogens provides novel insight on disease immunopathogenesis in this species and as a comparative model. Importantly, the data suggest that in some cases infection may be associated with subclinical immunopathologic perturbations that could impact overall individual and population health.

Analysis of Immune Response Markers in Jorge Lobo's Disease Lesions Suggests the Occurrence of Mixed T Helper Responses with the Dominance of Regulatory T Cell Activity.

Jorge Lobo's disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-ß and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-ß1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-ß1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-ß1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.

Nutritional status and immune response in murine experimental Jorge Lobo's disease.

There are no studies investigating the role of nutritional status and immunity associated with Jorge Lobo's disease. The objective of this study was to evaluate the effects of protein-calorie malnutrition on the immune response of BALB/c mice inoculated with Lacazia loboi. In this study,the animals were divided into four groups: G1: inoculated with restricted diet, G2: not inoculated with restricted diet, G3: inoculated with regular diet, G4: not inoculated with regular diet. The animals of groups G1 and G2 were submitted to malnutrition for 20 days and once installed the animals were inoculated intradermally into the footpad. After 4 months, they were euthanised for the isolation of peritoneal lavage cells and removal of the footpad. The production of IL-2, IL-4, IL-10, IL-12, IFN-γ, TNF-α, H2 O2 and nitric oxide (NO) was evaluated in the peritoneal lavage cells. The footpad was evaluated regarding the size of macroscopic lesions, number of fungi and viability index. The results showed that the infection did not exert great influence on the body weight of the mice and previous malnutrition was an unfavourable factor for viability index, number of fungi, macroscopic lesion size in the footpad and production of H2 O2 , NO, IL-12, IL-10 and IFN-γ, suggesting that malnutrition significantly altered fungal activity and peritoneal cells. The results suggest considerable interaction between nutrition and immunity in Jorge Lobo's disease.

Jorge Lobo's disease: immunohistochemical characterization of dendritic cells in cutaneous lesions.

BACKGROUND: Jorge Lobo's disease (JLD) is a cutaneous chronic mycosis caused by Lacazia loboi. We studied Factor XIIIa + dermal dendrocytes (FXIIIa + DD), Langerhans cells (LC) through the expression of langerin and the expression of S100 protein. METHODS: A total of 41 biopsies and 10 normal skins (control) were developed with a polymer-based immunohistochemical method. RESULTS: Lesions presented infiltrate comprising macrophages, some asteroid corpuscles, lymphocytes, multinucleated giant cells and a large number of fungi. LCs presented short dendrites and were scarcely distributed. Dermal langerin + cells were detected in nine JLD lesions. FXIIIa + DD were hypertrophic, visualized in the inflammatory infiltrate of JLD lesions. Cells S100+ were present in JLD and control group with a similar number of cells. A total of 14 specimens did not express FXIIIa, and this considerable number probably contributed to the statistical similarity with the control group. CONCLUSIONS: The results indicate that LCs are present in the immune response against Lacazia loboi. Some dermal langerin + cells could be another subset of dendritic cells. Our data indicate changes of LCs in JLD cutaneous lesions and present, for the first time, results that show langerin + cells in the dermis and corroborate previous observations on the participation of FXIIIa + DD in the in situ immune response in JLD.

Disseminated infection with Lacazia loboi and immunopathology of the lesional spectrum.

The pathogenesis of lacaziosis continues to be obscure, and works have investigated the blood systemic immune response or the dermal immune response in restricted lesions in different body regions. Some authors describe that the inflammatory infiltrate in lacaziosis lesions showed a predominance of macrophages followed by CD45RO(+), CD4(+), and CD8(+) T cells; CD57(+) natural killer cells; S-100(+) cells; and CD20(+) B lymphocytes. A 54-year-old man and living in the State of Para, Amazon region, Brazil, was seen with a lesion on the left lower limb, which had started as a small nodular area 18 years ago. The lesion showed progressive growth and disseminated to other parts of the body. Our findings showed that dermal immune response differs depending on the type of lesions and clinical presentation, with presence of CD1a(+), FXIIIa(+), CD45(+), CD4(+), CD8(+), and S-100(+) cells and cytokine profile with expression of interleukin 1 ß, tumor necrosis factor α, transforming growth factor ß, IL-10, and interferon γ.

Analysis of immune response markers in Jorge Lobo's disease lesions suggests the occurrence of mixed T Helper responses with the dominance of regulatory T cell activity

Jorge Lobo's disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-ß and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-ß1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-ß1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-ß1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.

Nutritional status and immune response in murine experimental Jorge Lobo's disease

There are no studies investigating the role of nutritional status and immunity associated with Jorge Lobo's disease. The objective of this study was to evaluate the effects of protein-calorie malnutrition on the immune response of BALB/c mice inoculated with Lacazia loboi. In this study,the animals were divided into four groups: G1: inoculated with restricted diet, G2: not inoculated with restricted diet, G3: inoculated with regular diet, G4: not inoculated with regular diet. The animals of groups G1 and G2 were submitted to malnutrition for 20 days and once installed the animals were inoculated intradermally into the footpad. After 4 months, they were euthanised for the isolation of peritoneal lavage cells and removal of the footpad. The production of IL-2, IL-4, IL-10, IL-12, IFN-γ, TNF-α, H2 O2 and nitric oxide (NO) was evaluated in the peritoneal lavage cells. The footpad was evaluated regarding the size of macroscopic lesions, number of fungi and viability index. The results showed that the infection did not exert great influence on the body weight of the mice and previous malnutrition was an unfavourable factor for viability index, number of fungi, macroscopic lesion size in the footpad and production of H2 O2 , NO, IL-12, IL-10 and IFN-γ, suggesting that malnutrition significantly altered fungal activity and peritoneal cells. The results suggest considerable interaction between nutrition and immunity in Jorge Lobo's disease

Plasmacytoid dendritic cells in cutaneous lesions of patients with chromoblastomycosis, lacaziosis, and paracoccidioidomycosis: a comparative analysis.

Plasmacytoid dendritic cells (pDCs) are characterized by expression of CD123 and BDCA-2 (Blood Dendritic Cell Antigen 2) (CD303) molecules, which are important in innate and adaptive immunity. Chromoblastomycosis (CBM), lacaziosis or Jorge Lobo's disease (JLD), and paracoccidioidomycosis (PCM), are noteworthy in Latin America due to the large number of reported cases. The severity of lesions is mainly determined by the host's immune status and in situ responses. The dendritic cells studied in these fungal diseases are of myeloid origin, such as Langerhans cells and dermal dendrocytes; to our knowledge, there are no data for pDCs. Forty-three biopsies from patients with CBM, 42 from those with JLD and 46 diagnosed with PCM, were evaluated by immunohistochemistry. Plasmacytoid cells immunostained with anti-CD123 and anti-CD303 were detected in 16 cases of CBM; in those stained with anti-CD123, 24 specimens were obtained from PCM. We did not detect the presence of pDCs in any specimen using either antibody in JLD. We believe that, albeit a secondary immune response in PCM and CBM, pDCs could act as a secondary source of important cytokines. The BDCA-2 (CD303) is a c-type lectin receptor involved in cell adhesion, capture, and processing of antigens. Through the expression of the c-lectin receptor, there could be an interaction with fungi, similar to other receptors of this type, namely, CD207 in PCM and CD205 and CD209 in other fungal infections. In JLD, the absence of expression of CD123 and CD303 seems to indicate that pDCs are not involved in the immune response.

Detection of cytokines and nitric oxide synthase in skin lesions of Jorge Lobo's disease patients

Studies investigating the immunopathological aspects of Jorge Lobo's disease have shown that the inflammatory infiltrate consists mainly of histiocytes and multinucleated giant cells involving numerous yeast-like cells of Lacazia loboi, with the T lymphocytes more common than B lymphocytes and plasma cells. The quantification of cytokines in peripheral blood mononuclear cells culture supernatant has revealed alterations in the cytokines profile, characterized by predominance of a Th2 profile. In view of these findings and of the role of cytokines in cell interactions, the objective of the present study was to investigate the presence of the cytokines IL-10, TGF-ß1 and TNF-α, as well as iNOS enzyme in granulomas induced by L. loboi. Histological sections obtained from skin lesions of 16 patients were analyzed by immunohistochemistry for the presence of these cytokines and iNOS. The results showed that TGF-ß1 was the cytokine most frequently expressed by cells present in the inflammatory infiltrate, followed by IL-10. There was a minimum to discrete positivity of cells expressing TNF-α and iNOS. The results suggest that the presence of immunosuppressive cytokines in skin lesions of patients with the mycosis might be responsible for the lack of containment of the pathogen as demonstrated by the presence of numerous fungi in the granuloma.

Pesquisa de imunoglobulinas e complemento no granuloma da doença de Jorge Lobo / Immunoglobulin and complement research in the granuloma of Jorge Lobo's disease

A doença de Jorge Lobo é micose cutânea-subcutânea de evolução crônica causada pelo fungo Lacazia loboi. Esta micose é predominante na região Amazônica e afeta, principalmente, trabalhadores rurais que vivem em contato constante com a vegetação e o solo, como é o caso dos seringueiros dessa região. Os estudos abordando os aspectos imunopatológicos desta micose são ainda muito escassos, possivelmente devido ao não cultivo do seu agente etiológico. Considerando que existem estudos demonstrando a participação da resposta imune humoral na doença de Jorge Lobo, onde os pacientes exibem níveis séricos elevados de imunoglobulinas e complemento, e alterações no perfil de citocinas representadas por um predomínio do perfil Th2, o presente estudo foi realizado com a finalidade de avaliar a presença de anticorpos e components do sistema complemento no granuloma induzido pelo L.loboi. Participaram destte estudo 11 pacientes portadores da micose, provenientes do estado do Acre, dos quais foram coletados fragmentos de pele das lesões. O material foi avaliado quanto à presença de imunoglobulinas e complemento, através da técnica de imunofluorescência e submetido à coloração pela hematoxilina-eosina para serem avaliados histopatologicamente. Os resultados obtidos revelaram depósitos dessas proteínas envolvendo a parede celular dos fungos em todos os pacientes avaliados e presença de um infiltrado inflamatório constituído predominantemente por histiócitos, células gigantes multinucleadas e numerosos fungos. Os linfócitos estavam presentes em número discreto a moderado e os plasmócitos, embora em número inferior, sempre estiveram presentes. Os resultados obtidos neste estudo revelam a presença de anticorpos e complemento no granuloma da doença de Jorge Lobo e sugerem a participação dessas proteínas nos mecanismos de defesa contra o fungo L. loboi.