Insulin effects on Paracoccidioides brasiliensis-induced systemic mycosis in healthy and diabetic mice

A paracoccidioidomicose é uma enfermidade sistêmica causada principalmente pelo fungo Paracoccidioides brasiliensis. Recentemente, observou-se que a capacidade fagocítica dos macrófagos alveolares (MA) em animais diabéticos está diminuída em comparação aos MA de animais sadios, e que a insulina estimula a atividade fagocítica em MA oriundos de animais diabéticos e sadios por mecanismos diferentes. Neste projeto, usando um modelo de carência relativa de insulina (diabetes mellitus experimental), estudamos a intervenção da insulina em um modelo de infecção sistêmica. Após aprovação do comitê de ética (protocolo CEUA/FCF/421), camundongos machos da linhagem C57BL/6 (diabéticos, 60 mg/kg aloxana/10 dias, e seus respectivos controles) receberam injeção intratraqueal contendo suspensão de leveduras de P. brasiliensis ou volume equivalente de PBS estéril. Animais dos grupos de tratamento receberam insulina pela via subcutânea diariamente por 12 dias. Nas amostras, foram avaliados: a) o número de células dos lavados peritoneal (LPe) e broncoalveolar (LBA), o leucograma, e a glicemia (monitor de glicose); b) os níveis séricos de insulina no soro pela técnica de ELISA; c) as concentrações de citocinas (TNF-α, IL-6, IL-4, IL-10, IL-12, CINC-1, CINC-2, CINC-3) nos LPe e LBA e nos homogenatos (ELISA). Após incubação de 55 dias, comparados aos controles (2.9±0,4g e 192±7.5 mg/dL), animais tornados diabéticos (0.87 ± 0,25 g e 570,1 ± 9,27mg/dL) apresentaram redução no ganho de massa corpórea durante o período de experimentação e elevados níveis de glicose sanguínea. O tratamento de insulina reduziu os níveis de glicose (547±36,8mg/dL vs.323,6±36,9mg/dL), embora não o suficiente para tornar os animais normoglicêmicos. Comparados aos controles, animais diabéticos apresentaram número reduzido de leucócitos no LPe (2.2 x106 ± 0.2cells/mm3 vs 1.3 x106 ± 0.1cells/mm3) e, no LBA, reduzidas concentrações de CINC-2 (662,3±73,8pg/mL vs 312,7±114,7pg/mL), CINC-1 (115,5.0±25,5pg/mL vs 88,3±24,7pg/mL) e IL-10(320,9±58,4pg/mL vs 161,0±59,4pg/mL) depois da infecção. O tratamento com insulina restaurou a concentração de leucócitos nos LPe de animais diabéticos, mas não no LBA. Os dados sugerem que a insulina modula a produção/liberação de citocinas, sem alterar a migração de leucócitos para LBA e restaurando a migração destes para LPe durante o curso da paracoccidioidomicose.Paracoccidioidomycosis is a systemic disease mainly caused by Paracoccidioides brasiliensis fungus that interact with antigen-presenting cells, changing its main biological functions. Recently it was observed that the phagocytic capacity of these cells in diabetic animals for IgG opsonized targets is decreased compared to healthy animals, and that insulin stimulates the phagocytic activity in alveolar macrophages in from diabetic and healthy animals, by different mechanisms. In this project, using a model of relative lack of insulin (experimental diabetes mellitus), we studied the intervention of insulin in a model of systemic infection. After approval by the committee of ethics (protocol CEUA/FCF/421), C57BL/6 male diabetic (60mg/kg aloxan/10days) mice and their respective controls were subjected to intratracheal injection of suspension of P. brasiliensis or an equivalent volume of TBS sterile. In the forty-third day, insulin-treated mice were treated subcutaneously daily with insulin for 12 days at 6 P.M. We evaluated: a) the number of cells of the peritoneal(PeL) and bronchoalveolar (BAL) fluids, the leucogram and the glucose levels; b) the levels of insulin on the serum by the technique of ELISA; c) the levels of cytokines (TNF-α, IL-6, IL-4, IL-10, IL-12, CINC-1, CINC-2, CINC-3) in the BAL and PeL fluid, and on organ homogenates. After 55 days, relative to controls (2.9±0,4g and 192±7.5 mg/dL)), mice rendered diabetic (0.87 ± 0,25 g and 570,1 ± 9,27mg/dL) exhibited a reduction in body weight gain during the experimental period and sharply elevated blood glucose levels. Treatment of diabetic animals with insulin induced a reduction in blood glucose levels (547±36,8mg/dL vs.323,6±36,9mg/dL), but it was not sufficient to reduce glycemia to control values. In addition, relative to controls, infected diabetic mice exhibited a reduction in the number of leukocytes into the PeL fluid ((2.2 x106 ± 0.2cells/mm3 vs 1.3 x106 ± 0.1cells/mm3) and reduced BAL concentrations of CINC-2 (662,3±73,8pg/mL vs 312,7±114,7pg/mL), CINC-1 (115,5.0±25,5pg/mL vs 88,3±24,7pg/mL) and IL-10 (320,9±58,4pg/mL vs 161,0±59,4pg/mL) after P. brasiliensis infection. Treatment of diabetic mice with insulin restored concentrations of leukocytes in the PeL fluid but not in the BAL. Data presented suggest that insulin modulates the production/release of cytokines but not leukocyte migration to the BAL while restoring this paramether on the PeL during the course of P. brasiliensis fungus-induced PCM

Insulin modulates cell migration and cytokines secretion during pulmonary infection caused by Paracoccidioides brasiliensis in diabetic and non-diabetic mice

Diabetes mellitus (DM) compreende um conjunto de doenças metabólicas de grande importância e incidência mundial. Nele, o DM do tipo 1 é caracterizado pela destruição de células pancreáticas produtoras de insulina, e dentre seus sintomas, a disfunção imunológica relacionada à falta de insulina foi observada por diversos estudos, descrevendo pacientes diabéticos como mais susceptíveis a infecções e complicações decorrentes destas. Paracoccidioidomicose (PCM) é uma enfermidade sistêmica causada por fungos da espécie Paracoccidioides sp., bastante importante no Brasil e endêmica em toda a América Latina. Este trabalho utiliza um modelo de carência relativa de insulina (DM experimental) para estudar a intervenção da insulina em um modelo de micose pulmonar causada por P. brasiliensis, analisando o processo de migração celular (expressão de moléculas de adesão por imunohistoquímica e fenótipo dos leucócitos do pulmão por citometria de fluxo), os mecanismos moleculares (produção/liberação de citocinas por cytometric bead array), intracelulares (vias de sinalização por Western blot), e a atividade fagocítica e microbicida dos macrófagos alveolares. Em resultados observamos que, comparados aos não-diabéticos, camundongos tornados diabéticos apresentam maior susceptibilidade evidenciada por menor atividade fagocítica e reduzidas secreções de interferon-γ e de interleucina-12 na fase inicial da inflamação, que leva a uma resposta menos efetiva com menor expressão de molécula de adesão de células vasculares, reduzidas populações de linfócitos TCD4+, TCD8+, células natural killer, culminando em inflamação crônica resultante da proliferação aumentada do fungo nos pulmões (aumento de interferon-γ e fator necrótico tumoral-β). Vemos ainda que o tratamento de insulina em animais diabéticos restaurou as secreções de citocinas pró-inflamatórias e a atividade fagocítica de macrófagos em 24 horas de infecção, e aumentou a celularidade, a expressão de moléculas de adesão de células vasculares-1 e restaurou as populações de linfócitos B, de células natural killer e de células coestimuladas por CD80, além de reduzir a inflamação crônica no pulmão. Estes dados em conjunto nos permitem inferir que a insulina modulou o ambiente inflamatório de animais tornados diabéticos de formas diferentes em estágios iniciais e tardios da infecção pelo isolado Pb18 do Paracoccidioides brasiliensisDiabetes mellitus comprehends a group of metabolic diseases of great importance and incidence worldwide. Type 1 diabetes mellitus is characterized by destruction of insulin producing-pancreatic cells and, among its symptoms, an impaired immunological function has been observed in many studies having diabetic patients described as more susceptible to infections and complications resulted of them. Paracoccidioidomycosis is a systemic disease caused by fungi of Paracoccidioides spp. , also of great importance in Brazil and endemic in the whole Latin America. This work uses a model of experimental T1DM to investigate the intervention of insulin in a model of murine PCM induced by Paracoccidioides brasiliensis, analyzing the process of cell migration (adhesion molecules expression, leukocyte phenotyping), molecular mechanisms (production and secretion of cytokines), intracellular mechanisms (signaling pathways) and phagocytic and microbicidal activities in alveolar macrophages. In results, compared to controls, we observed higher susceptibility in diabetic mice to PCM, evidenced by reduced phagocytic activity and reduced levels of interferon-γ and interleukin-12 on initial stages of infection, and a less effective inflammation with lesser expression of adhesion molecules, reduced migration of TCD4+, TCD8+, NK cells and B lymphocytes, resulting in chronic inflammation caused by higher fungal proliferation in lungs (higher interferon-γ and tumours necrosis factor-α levels). In addition, we saw treatment with insulin in diabetic animals restored secretion of pro-inflammatory cytokines and phagocytic activity on early stages and allowed higher cellularity, higher expression of vascular cells adhesion molecule-1 and restored populations of B lymphocytes, NK cells and the expression of costimularoty molecule CD80, also reducing the chronic inflammation in lungs. Taken together, these data lead us to suggest insulin modulated the inflammatory microenvironment in lungs of mice rendered diabetic, in different forms on earlier and later stages of an infection by Pb18 isolat

Genetic deficiency of indoleamine 2,3-dioxygenase aggravates vascular but not liver disease in a nonalcoholic steatohepatitis and atherosclerosis comorbidity model

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that increases cardiovascular disease risk. Indoleamine 2,3-dioxygenase-1 (IDO1)-mediated tryptophan (Trp) metabolism has been proposed to play an immunomodulatory role in several diseases. The potential of IDO1 to be a link between NASH and cardiovascular disease has never been investigated. Using Apoe−/− and Apoe−/−Ido1−/− mice that were fed a high-fat, high-cholesterol diet (HFCD) to simultaneously induce NASH and atherosclerosis, we found that Ido1 deficiency significantly accelerated atherosclerosis after 7 weeks. Surprisingly, Apoe−/−Ido1−/− mice did not present a more aggressive NASH phenotype, including hepatic lipid deposition, release of liver enzymes, and histopathological parameters. As expected, a lower L-kynurenine/Trp (Kyn/Trp) ratio was found in the plasma and arteries of Apoe−/−Ido1−/− mice compared to controls. However, no difference in the hepatic Kyn/Trp ratio was found between the groups. Hepatic transcript analyses revealed that HFCD induced a temporal increase in tryptophan 2,3-dioxygenase (Tdo2) mRNA, indicating an alternative manner to maintain Trp degradation during NASH development in both Apoe−/− and Apoe−/−Ido1−/mice−. Using HepG2 hepatoma cell and THP1 macrophage cultures, we found that iron, TDO2, and Trp degradation may act as important mediators of cross-communication between hepatocytes and macrophages regulating liver inflammation. In conclusion, we show that Ido1 deficiency aggravates atherosclerosis, but not liver disease, in a newly established NASH and atherosclerosis comorbidity model. Our data indicate that the overexpression of TDO2 is an important mechanism that helps in balancing the kynurenine pathway and inflammation in the liver, but not in the artery wall, which likely determined disease outcome in these two target tissues

Insulin Modulates Paracoccidioides brasiliensis-Induced Inflammation by Restoring the Populations of NK Cells, Dendritic Cells, and B Lymphocytes in Lungs

Paracoccidioidomycosis, a key issue for Brazilian health service, can be aggravated in patients with impaired immunological responses, such as diabetic patients. We evaluated the role of insulin in inflammatory parameters in diabetic and nondiabetic mice using a systemic mycosis Paracoccidioides brasiliensis (Pb) model. Diabetic C57BL-6 mice and controls were infected with Pb18 and treated with insulin for 12 days prior to experiments. After 55 days, infected diabetic mice exhibited fewer leukocytes in both peritoneal lavage fluid (PeLF) and bronchoalveolar lavage fluid and reduced secretion of interleukin- (IL-) 6 in lungs. In addition, diabetic mice presented a reduced influx of TCD4+ cells, TCD8+ cells, B lymphocytes, NK cells, and dendritic cells compared to control infected groups. Insulin treatment restored the leukocyte number in PeLF and restored the presence of B lymphocytes, dendritic cells, and NK cells in lungs of diabetic animals. The data suggest that diabetic mice present impaired immunological response to Pb18 infection and insulin modulates inflammation by reducing IL-6 levels in lung and CINC-1 levels in spleen and liver homogenates, restoring leukocyte concentrations in PeLF and also restoring populations of dendritic cells and B lymphocytes in lungs of diabetic mice, permitting the host to better control the infection