Chronic exposure to cigarette smoke transiently worsens the disease course in a mouse model of pulmonary paracoccidioidomycosis

Paracoccidioidomycosis (PCM) may present as an acute/subacute clinical form, characterized by a progressive disease arising from the airborne initial infection, or, most often, as an asymptomatic or subclinical infection that may manifest later during an individual’s life, the chronic form. Epidemiological studies show the existence of a strong association between smoking and the development of the chronic form. Current evidence demonstrates that cigarette smoke (CS) has immunosuppressive properties that could be implicated in the increasing susceptibility to the chronic form of PCM. To address this issue, we developed a murine model of a non-progressive pulmonary form of PCM that was exposed to CS at a magnitude that mimicked a moderate smoker. The chronic CS exposure started after 2 weeks and lasted up until 20 weeks post-infection, with the aim of mimicking human natural history, since it is estimated that individuals from endemic areas are infected early in life. The control group consisted of infected but not CS-exposed mice. We assessed the lung fungal burden (colony forming units [CFU]) and the area affected by the granulomatous inflammatory response, fungal dissemination to spleen and liver, and, by immunohistochemistry, the presence of CD4 and CD8 lymphocytes, CD68 and MAC-2 macrophages, and IFN-γ, IL-10 and TNF expressing cells within the granulomatous response. We detected a CS effect as early as 2 weeks after exposure (four weeks post-infection) when the lung CFU of exposed animals was significantly higher than in their non-exposed counterparts. At 12 weeks, the CS-exposed animals presented a more severe disease, as witnessed by the persistent higher lung fungal load (although it did not reach statistical significance [ p = 0.054]), greater dissemination to other organs, greater affected area of the lung, decreased IFN-γ/IL-10 ratio, and higher TNF expression within the granulomas, compared with CS-non-exposed mice. The number of CD4 and CD8 lymphocytes infiltrating the granulomas was similar between both mice groups, but there was a decrease in the number of MAC-2+ macrophages. No difference was noted in the CD68+ macrophage number. However, the follow-up in week 20 showed that the immunological effects of exposure to CS ceased, with both CS and NCS mice showing the same infectious features, i.e., a trend for resolution of the infection. In conclusion, we show that chronic CS-exposure alters the course of the disease in an experimental model of subclinical pulmonary PCM, confirming the epidemiological link between CS-exposure and the chronic form of PCM. However, we also show that this effect is transitory, being detected between 4- and 12-weeks post-infection but not thereafter. The possible immune mechanisms that mediate this effect and the reasons for its transitory effect are discussed

Use of dendritic cells pulsed with peptide 10 (P10) Paracoccidioidesbrasiliensis to control experimental paracoccidioidomycosis. Reversal of the anergic state, association with antifungal and control acute infection

A paracoccidioidomicose (PCM) é uma micose sistêmica e endêmica na América Latina com maior prevalência no Brasil, Colômbia e Venezuela. A doença é causada pelos fungos P. brasiliensis e P. lutzii. As células dendríticas são eficientes apresentadoras de antígenos e quando utilizadas como adjuvante podem ser de 100 a 1000 vezes mais efetiva nesta função. O peptídeo P10 corresponde a um trecho específico de 15 aminoácidos derivado da gp43, principal antígeno diagnóstico, e é reconhecido pelos linfócitos T CD4+ capaz de induzir resposta preferencialmentedo tipo Th1 e conferindo proteção no modelo experimental. Células indiferenciadas foram obtidas a partir de medula óssea de camundongos machos BALB/c B10.A, cultivadas na presença de GM-CSF e IL-4 por 9 dias, para a diferenciação de células dendríticas (DC). As células foram incubadas na presença do P10 por 2 horas, em estufa com 5% de CO2 a 37°C, e foram utilizadas nas imunizações. A maturação das células foi observada por citometria de fluxo com os marcadores CD11c, MHC-II, CD80 e CD86. Camundongos da linhagem BALB/c e B10.A foram submetidos à imunossupressão com dexametasona, por 20 dias, seguido pela infecção intratraqueal com o isolado Pb18. Após 30 dias, os grupos foram tratados com DCs ou DCs pulsadas com P10 em associação ou não com sulfametoxazol/Trimetoprim, por um período de 15 dias. O sacrifício ocorreu 45 dias após a infecção e foram retirados os pulmões, baço e fígado para quantificação de carga fúngica, análise histológica e dosagem de citocinas. Observamos diminuição significativa da carga fúngica nos pulmões dos animais que receberam DCs pulsadas com P10 associada ou não ao tratamento medicamentoso. Não foi observado crescimento fúngico em outros órgãos como fígado e baço em ambos os grupos. A análise histológica revelou redução da carga fúngica e preservação do parênquima pulmonar nos grupos tratados com Dcs pulsadas com P10. As dosagens das citocinas indicaram uma resposta imune mista Th1/Th2. Inicialmente reportamos que as DCs pulsadas com P10 reduz a carga fúngica em camundongos infectados. Neste trabalho, reportamos que as DCs pulsadas com P10 podem também reduzir a carga fúngica em animais anérgicos, mimetizando pacientes com a forma aguda/subaguda da doençaParacoccidioidomycosis (PCM) is a systemic and endemic mycosis in South America, with higher prevalence in Brazil, Colombia and Venezuela. This disease is caused by fungi P. brasiliensis and P. lutzii. The peptide P10 matches a specific path of 15 amonoacids which is derived from gp43, main diagnostic antigen. This peptide is recognized by the T CD4+ lymphocytes and induces a response type Th1, giving protection at an experimental mode. Some dendritic cells (DC) have an efficient antigen and, when used as adjuvant, they can be 100 to 1000 times more effective. Undifferentiated cells were obtained from bone marrow of male mice type BALB/c and B10.A and cultivated in the presence of GM-CSF and IL-4 for 9 days, so that DCs would be differentiated. The cells were incubated in the presence of P10 for 2 hours in incubator at 37°C with 5% of CO2, and later utilized in the immunizations. Maturation of the cells was observed by flow cytometry with CD11c, MHC-II, CD80 and CD86 markers. Mice types BALB/c and B10.A had been submitted to an immunosuppression with dexamethasone for 20 days, before being intratracheally infected with isolate Pb18. After 30 days, the group of animals received immunizations with DCs or DCs pulsed with P10 associated or not with the treatment by Sulfametoxazol/Trimetoprim, this for a period of 15 days. The sacrifice occurred 45 day after the animals had been infected, and their lungs, spleen and liver were taken out for quantification of fungal burden, histology and cytokine assay. We observed a significant decrease of fungal burden in the lungs of animal that received DC pulsed with P10, associated or not with the drug treatment. In both groups we didn\'t observe fungal growth in organs such as liver and spleen. The histological analyses showed reduced fungal burden and preservation of lung parenchyma. The dosages of cytokines showed an immune response mixed Th1/Th2 type. At first we reported that the DCs pulsed with P10 were responsible for reducing the fungal burden in infected mice. In this paper we report that the DCs pulsed with P10 may also be responsible for reducing fungal burden in anergic animals, mimicking patients with the acute/subacute form of the diseas

Characterization of the modulating activity of differentiated dendritic cell from monocytes isolated from mice infected with virulent isolates of Paracoccidioides spp pulsed with the peptide P10 in the treatment of Paracoccidiodomycosis

Paracoccidioidomicose (PCM), micose sistêmica prevalente na América Latina, é uma doença granulomatosa causada pelo fungo termodimórfico do gênero Paracoccidioides spp. O maior número de casos dessa doença tem sido reportado no Brasil, Colômbia, Venezuela e Argentina. A existência de extensas áreas endêmicas, alto potencial incapacitante devido a fibroses pulmonares, grande quantidade de mortes prematuras, faz com que a PCM seja considerada um grave problema de Saúde Pública. Apesar do tratamento medicamentoso ser relativamente eficiente, o tempo longo de uso das drogas e os efeitos colaterais provocados podem levar a diminuição da adesão ao tratamento diminuindo assim eficácia. O uso de vacinas terapêuticas pode ser uma importante ferramenta no controle da PCM. Uma das modalidades investigadas pelo nosso grupo é a utilização de células dendríticas (DCs) pulsadas com o peptídeo P10 como vacina terapêutica no controle da PCM. Resultados apresentados por nosso grupo, demonstraram que DCs diferenciadas a partir de células de medula óssea de camundongos saudáveis pulsadas com o peptídeo 10 (P10) podem ser utilizadas como adjuvante no tratamento da paracoccidioidomicose experimental em camundongos imunocompetentes e imunossuprimidos. Assim sendo, nossa proposta no presente trabalho foi avaliar se DCs diferenciadas de células de medula óssea (BMDCs) ou de monócitos circulantes (MoDCs) de camundongos (BALB/c) infectados apresentam modulação prévia devido à exposição a antígenos fúngicos, avaliamos in vitro a capacidade das células dendríticas diferenciadas a partir de monócitos circulantes de camundongos infectados com Pb18 estimular proliferação de linfócitos CD8+ e CD4+, analisamos a capacidade de células dendríticas derivadas de monócitos circulantes de camundongos infectados com Pb18 pulsadas com P10 em induzir uma resposta protetora. Nossos resultados obtidos são promissores e reforçam dados já publicados pelo grupo os quais demonstraram a eficiência do peptídeo P10 no tratamento da paracoccidioidomicose experimental; apontam que o P10 é capaz de modular ativamente células dendríticas diferenciadas de monócitos circulantes e diferenciadas de células de medula óssea; demonstram que BMDCs e MoDCs pulsadas ou não com P10 tem a capacidade de estimular proliferação de linfócitos T CD4+ e CD8+; confirmaram que BMDC e MoDCs pulsadas ou não com P10 diminuem a carga fúngica no tecido pulmonar, estimulando um padrão de citocinas misto, porém com predominância de citocinas pró-inflamatórias que incitam resposta imune celular do tipo Th1, que é protetora na PCM. Estes dados foram respaldados com a análise dos cortes histológicos onde os pulmões dos camundongos tratados com BMDCs ou MoDCs pulsadas com P10 apresentaram parênquima pulmonar mais conservado em comparação com os pulmões dos grupos que não receberam tratamento com BMDCs ou MoDCs, com diminuição significativa de células fúngicas viáveisParacoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America, is a granulomatous disease caused by the thermodymorphic fungus of the genus Paracoccidioides spp. The highest number of cases of this disease has been reported in Brazil, Colombia, Venezuela and Argentina. The existence of extensive endemic areas, a high incapacitating potential due to pulmonary fibroses, and a large number of premature deaths, makes the PCM considered a serious public health problem. Although the drug treatment is relatively efficient, the long time of use of the drugs and the side effects provoked can lead to the decrease of the adherence to the treatment thus diminishing their effectiveness. The use of therapeutic vaccines can be an important tool in the control of PCM. One of the modalities investigated by our group is the use of dendritic cells (DCs) pulsed with the peptide P10 as therapeutic vaccine in the control of PCM. Results presented by our group demonstrated that DCs differentiated from bone marrow cells of healthy mice pulsed with peptide 10 (P10) can be used as an adjuvant in the treatment of experimental paracoccidioidomycosis in immunocompetent and immunosuppressed mice. Thus, our proposal in the present study evaluated the capacity of differentiated DCs of bone marrow cells (BMDCs) or circulating monocytes (MoDCs) of infected mice (BALB/c) exhibited previous modulation due to exposure to fungal antigens, we evaluated in vitro the ability of differentiated dendritic cells from circulating monocytes from Pb18 infected mice to stimulate proliferation of CD8+ and CD4+ lymphocytes, we analyzed the ability of dendritic cells derived from circulating monocytes from mice infected with P. brasiliensis pulsed with P10 to induce a protective response. Our results are promising and reinforce data already published by the group which demonstrated the efficiency of the peptide P10 in the treatment of experimental paracoccidioidomycosis; suggest that P10 is capable of actively modulating differentiated dendritic cells from circulating and differentiated monocytes from bone marrow cells; demonstrate that BMDCs and MoDCs pulsed or not with P10 have the ability to stimulate proliferation of CD4+ and CD8+ T lymphocytes; confirmed that BMDC and MoDCs pulsed or not with P10 decrease the fungal load in lung tissue, stimulating a pattern of mixed cytokines but with a predominance of pro-inflammatory cytokines that stimulate Th1-type cellular immune response that is protective in PCM, these data were supported with analysis of the histological sections where the lungs of the mice treated with BMDCs or MoDCs pulsed or not with P10 showed more conserved pulmonary parenchyma compared to the lungs of the groups that did not receive treatment with BMDCs or MoDCs, with significant decrease of viable fungal cell

In Vitro and In Vivo Effect of Peptides Derived from 14-3-3 <i>Paracoccidioides</i> spp. Protein

Background: Paracoccidioidomycosis (PCM) is a chronic disease that causes sequelae and requires prolonged treatment; therefore, new therapeutic approaches are necessary. In view of this, three peptides from Paracoccidioides brasiliensis 14-3-3 protein were selected based on its immunogenicity and therapeutic potential. Methods: The in vitro antifungal activity and cytotoxicity of the 14-3-3 peptides were evaluated. The influence of the peptides in immunological and survival aspects was evaluated in vivo, using Galleria mellonella and the expression of antimicrobial peptide genes in Caenorhabditis elegans. Results: None of the peptides were toxic to HaCaT (skin keratinocyte), MRC-5 (lung fibroblast), and A549 (pneumocyte) cell lines, and only P1 exhibited antifungal activity against Paracoccidioides spp. The peptides could induce an immune response in G. mellonella. Moreover, the peptides caused a delay in the death of Paracoccidioides spp. infected larvae. Regarding C. elegans, the three peptides were able to increase the expression of the antimicrobial peptides. These peptides had essential effects on different aspects of Paracoccidioides spp. infection showing potential for a therapeutic vaccine. Future studies using mammalian methods are necessary to validate our findings

In Vitro and In Vivo Effect of Peptides Derived from 14-3-3 Paracoccidioides spp. Protein

Background: Paracoccidioidomycosis (PCM) is a chronic disease that causes sequelae and requires prolonged treatment; therefore, new therapeutic approaches are necessary. In view of this, three peptides from Paracoccidioides brasiliensis 14-3-3 protein were selected based on its immunogenicity and therapeutic potential. Methods: The in vitro antifungal activity and cytotoxicity of the 14-3-3 peptides were evaluated. The influence of the peptides in immunological and survival aspects was evaluated in vivo, using Galleria mellonella and the expression of antimicrobial peptide genes in Caenorhabditis elegans. Results: None of the peptides were toxic to HaCaT (skin keratinocyte), MRC-5 (lung fibroblast), and A549 (pneumocyte) cell lines, and only P1 exhibited antifungal activity against Paracoccidioides spp. The peptides could induce an immune response in G. mellonella. Moreover, the peptides caused a delay in the death of Paracoccidioides spp. infected larvae. Regarding C. elegans, the three peptides were able to increase the expression of the antimicrobial peptides. These peptides had essential effects on different aspects of Paracoccidioides spp. infection showing potential for a therapeutic vaccine. Future studies using mammalian methods are necessary to validate our findings