Activation of monocytes and cytokine production in patients with peripheral atherosclerosis obliterans

BACKGROUND: Arterial peripheral disease is a condition caused by the blocked blood flow resulting from arterial cholesterol deposits within the arms, legs and aorta. Studies have shown that macrophages in atherosclerotic plaque are highly activated, which makes these cells important antigen-presenting cells that develop a specific immune response, in which LDLox is the inducing antigen. As functional changes of cells which participate in the atherogenesis process may occur in the peripheral blood, the objectives of the present study were to evaluate plasma levels of anti-inflammatory and inflammatory cytokines including TNF-α, IFN-γ, interleukin-6 (IL-6), IL-10 and TGF-β in patients with peripheral arteriosclerosis obliterans, to assess the monocyte activation level in peripheral blood through the ability of these cells to release hydrogen peroxide (H(2)O(2)) and to develop fungicidal activity against Candida albicans (C. albicans) in vitro. METHODS: TNF-α, IFN-γ, IL-6, IL-10 and TGF-β from plasma of patients were detected by ELISA. Monocyte cultures activated in vitro with TNF-alpha and IFN-gamma were evaluated by fungicidal activity against C. albicans by culture plating and Colony Forming Unit (CFU) recovery, and by H(2)O(2 )production. RESULTS: Plasma levels of all cytokines were significantly higher in patients compared to those detected in control subjects. Control group monocytes did not release substantial levels of H(2)O(2 )in vitro, but these levels were significantly increased after activation with IFN-γ and TNF-α. Monocytes of patients, before and after activation, responded less than those of control subjects. Similar results were found when fungicidal activity was evaluated. The results seen in patients were always significantly smaller than among control subjects. Conclusions: The results revealed an unresponsiveness of patient monocytes in vitro probably due to the high activation process occurring in vivo as corroborated by high plasma cytokine levels

Chloroquine is therapeutic in murine experimental model of paracoccidioidomycosis

Chloroquine, due to its basic properties, has been shown to prevent the release of iron from holotransferrin, thereby interfering with normal iron metabolism in a variety of cell types. We have studied the effects of chloroquine on the evolution of experimental paracoccidioidomycosis by evaluating the viable fungal recovery from lung, liver and spleen from infected mice and H2O2, NO production, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-10 levels and transferrin receptor (TfR) expression from uninfected and infected peritoneal macrophages. Chloroquine caused a significant decrease in the viable fungal recovery from all organs tested, during all periods of evaluation. Peritoneal macrophages from chloroquine-treated infected mice showed higher H2O2 production and TfR expression, and decreased levels of NO, endogenous and stimulated-TNF-alpha, IL-6 and IL-10 during the three evaluated periods. However, despite its suppressor effects on the macrophage function, the chloroquine therapeutic effect upon murine paracoccidioidomycosis was probably due to its effect on iron metabolism, blocking iron uptake by cells, and consequently restricting iron to fungus growth and survival

Paracoccidioides brasiliensis interferes on dendritic cells maturation by inhibiting PGE(2) production

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil, whose etiologic agent is the thermodimorphic fungus of the genus Paracoccidioides, comprising cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii. The mechanisms involved in the initial interaction of the fungus with cells of the innate immune response, as dendritic cells (DCs), deserve to be studied. Prostaglandins (PGs) are eicosanoids that play an important role in modulating functions of immune cells including DCs. Here we found that human immature DCs derived from the differentiation of monocytes cultured with GM-CSF and IL-4 release substantial concentrations of PGE2, which, however, were significantly inhibited after challenge with P. brasiliensis. In vitro blocking of pattern recognition receptors (PRRs) by monoclonal antibodies showed the involvement of mannose receptor (MR) in PGE2 inhibition by the fungus. In addition, phenotyping assays showed that after challenge with the fungus, DCs do not change their phenotype of immature cells to mature ones, as well as do not produce IL-12 p70 or adequate concentrations of TNF-alpha. Assays using exogenous PGE2 confirmed an association between PGE2 inhibition and failure of cells to phenotypically mature in response to P. brasiliensis. We conclude that a P. brasiliensis evasion mechanism exists associated to a dysregulation on DC maturation. These findings may provide novel information for the understanding of the complex interplay between the host and this fungus.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

TNF-α production by DCs challenged with Pb18 or Pb265 or activated by LPS for 48 h, and measured by ELISA.

<p>The results are expressed as mean ± SD of independent experiments performed with cells obtained from 4 subjects. Statistically significant differences between groups are indicated: *p< 0.05.</p

Involvement of MR, TLR2, Dectin-1 and DC-SIGN on PGE₂ production inhibition induced by <i>P</i>. <i>brasiliensis</i> in human DCs.

<p>Cells were incubated with anti-MR, anti-TLR2, anti-Dectin-1 and/or anti-DC-SIGN monoclonal antibodies for 1 h, challenged with Pb18 or Pb265 (DCs/yeast ratio 5:1) for 4 and 24 h, and evaluated for PGE₂ production by ELISA. The results are expressed in mean ± SD of independent experiments performed with cells obtained from 4 subjects. Statistically significant differences between groups are indicated: *p< 0.05 versus control DCs and other available receptors.</p

Effect of exogenous PGE₂ on TNF-α production by DCs challenged with Pb18 or Pb265 by 48 h and evaluated by ELISA.

<p>The results are expressed in mean ± SD of experiments performed with cells obtained from 4 subjects. Statistically significant differences between groups are indicated: *p< 0.05 versus without PGE₂.</p

Human neutrophils produce IL-12, IL-10, PGE2 and LTB4 in response to Paracoccidioides brasiliensis. Involvement of TLR2, mannose receptor and dectin-1

The functions of phagocytic cells against pathogens are initiated by the interaction between membrane receptors and molecular structures which compose the cell wall of these microorganisms. Thus our study aimed to identify the neutrophil receptors involved in the recognition of different strains of Paracoccidioides brasiliensis and the consequent modulation of immune response through the production of cytokines and inflammatory mediators. Neutrophils did not produce TNF-alfa in response to both strains. However, these cells produce IL-12, mainly in tesponse to Pb 265, with participation of TLR2 and dectin-1. These cells also produce L-10, whose levels were higher for Pb 18 with involvement of TLR2 and MR and only TLR2 for Pb 265. The production of PGE2 and LTB4 was detected similarly for the two strains. For PGE2, MR and dectin-1 were involved, while in relation to LTB4, none of them. In summary, we demonstrated that neutrophils have a dynamic role during host immune response to P. brasiliensis, since in addition to their role as effector cells of innate immunity; they have the capacity to modulate innate and adaptative immune response against this fungus by producing cytokines and lipidic mediators. This modulation may be toward a pro- or anti-inflammatory pattern in a dependence of P. brasiliensis strains and PRR involved in fungus recognition by these cells. (C) 2014 Elsevier Ltd. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

PGE₂ production by DCs activated with LPS or challenged with Pb18 or Pb265 (DCs/Pb ratio 5:1) for different periods.

<p>The results are expressed in mean ± SD of experiments performed with cells from 5 subjects. Statistically significant differences between groups in the same period are indicated: *p< 0.05; ** p< 0.01; ***p<0.001 versus control DC; o p< 0.05 versus Pb18.</p

Effect of exogenous PGE₂ on percentage of cells, mean of fluorescence intensity (MFI) relative to the expression of CD40, CD80 (A) CD83, CD86 (B), HLA-DR, CXCR4 (C) CCR5, and CCR7 (D) by DCs after challenge or not with Pb18 and Pb265.

<p>The results are expressed in mean ± SD of experiments performed with cells obtained from 4 subjects. Statistically significant differences between groups are indicated: *p< 0.05 versus without PGE₂.</p