The Leukotriene B-4/BLT1 Axis Is a Key Determinant in Susceptibility and Resistance to Histoplasmosis

The bioactive lipid mediator leukotriene B-4 (LTB4) greatly enhances phagocyte antimicrobial functions against a myriad of pathogens. In murine histoplasmosis, inhibition of the LT-generating enzyme 5-lypoxigenase (5-LO) increases the susceptibility of the host to infection. In this study, we investigated whether murine resistance or susceptibility to Histoplasma capsulatum infection is associated with leukotriene production and an enhancement of in vivo and/or in vitro antimicrobial effector function. We show that susceptible C57BL/6 mice exhibit a higher fungal burden in the lung and spleen, increased mortality, lower expression levels of 5-LO and leukotriene B-4 receptor 1 (BLT1) and decreased LTB4 production compared to the resistant 129/Sv mice. Moreover, we demonstrate that endogenous and exogenous LTs are required for the optimal phagocytosis of H. capsulatum by macrophages from both murine strains, although C57BL/6 macrophages are more sensitive to the effects of LTB4 than 129/Sv macrophages. Therefore, our results provide novel evidence that LTB4 production and BLT1 signaling are required for a histoplasmosis-resistant phenotype.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

BLT₁ expression on C57BL/6 (A) and 129/Sv (B) macrophages.

<p>The resident cells were obtained as described in the Material and Methods, and the expression of the higher affinity receptor for LTB₄ was evaluated by flow cytometry. The mononuclear population was gated using the forward/side scatters and analyzed to determine the fluorescence intensity on the cells. The numbers in the histograms indicate the percentage of cells expressing the BLT₁ receptor. The results shown are from one experiment and are representative of two independent experiments.</p

Effect of exogenous LTB₄ on the phagocytosis of yeast by C57BL/6 and 129/Sv macrophages.

<p>PMs from C57BL/6 (A) and 129/Sv (B) mice were incubated for 2 h with IgG-opsonized or non-opsonized yeast at a yeast-to-cell ratio of 1∶5 in the presence or absence of exogenous LTB₄. The data are expressed as the mean ± SEM from one representative experiment of a total of two experiments (n = 3 to 5). *sv129 compared with C57BL/6; ^#129/Sv and C57BL/6 compared to LTB₄ treatment. p<0.05 was considered significant.</p

Differential leukocyte recruitment to the lung in resistant (129/Sv) and susceptible (C57BL/6) mice.

<p>Cells were obtained from mice at 7 and 14 days after i.t. injection of PBS or 5×10⁵<i>H. capsulatum</i> yeast cells, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085083#s3" target="_blank">Materials and Methods</a> section. The total cells (A), neutrophils (B) and mononuclear cells (C) were enumerated and identified after Rosenfeld staining. The data are expressed as the mean ± SEM from one representative experiment of a total of three experiments (n = 6/each experiment). *C57BL/6 and sv129 compared with PBS; ^#C57BL/6 compared with sv129. p<0.05 was considered significant.</p

TNF-α production in lung of resistant (129/Sv) and susceptible (C57BL/6) mice.

<p>Lungs were removed at 7 and 14 days after i.t. injection of PBS or 5×10⁵<i>H. capsulatum</i> yeast cells. TNF-α levels were determinate by ELISA. Data are presented as the mean ± SEM and are representative from one of two independent experiments (n = 6/each experiment). * 129/Sv compared with C57BL/6; ^#129/Sv <i>H. capsulatum</i> compared with C57BL/6 <i>H. capsulatum</i>. p<0.05 vs. PBS.</p

Differential 5-LO enzyme expression and LTB₄ production in C57BL/6 and 129/Sv mice.

<p>The expression of <i>alox5</i>, <i>aloxp5</i> and <i>Ltbr1</i> mRNA in PMs from C57BL/6 and sv129 (A) and PMs infected with <i>H. capsulatum</i> (B) for 6 h as described in the Material and Methods. (B) LTB₄ production by PMs from C57BL/6 and 129/Sv after <i>in vitro</i> infection with <i>H. capsulatum</i> (MOI = 1∶5) was measured by ELISA. The data are expressed as the mean ± SEM from one representative experiment of a total of two experiments (n = 3 to 5/each experiment). *sv129 compared with C57BL/6. p<0.05 was considered significant.</p

Survival rate and fungal burden in <i>H. capsulatum</i>-infected C57BL/6 and 129/Sv mice.

<p>C57BL/6 and sv129 mice were infected i.t. with 1×10⁶ (A) or 5×10⁵ (B) yeast cells, and their survival was followed for 60 days (n = 6). The fungal loads in the lungs (C) and spleen (D) of mice infected with 5×10⁵ yeast cells were evaluated at 7 and 14 days post-<i>H. capsulatum</i> infection. The data are expressed as the mean ± SEM from one representative experiment of a total of three experiments (n = 6/each experiment). *sv129 compared with C57BL/6. p<0.05 was considered significant.</p

Effect of endogenous and exogenous LTB₄ on the phagocytosis of <i>H. capsulatum</i> by C57BL/6 and 129/Sv macrophages.

<p>PMs from C57BL/6 and 129/Sv mice were incubated for 2 h with IgG-opsonized or non-opsonized yeast cells at a yeast-to-cell ratio of 1∶5. (A) PMs were pretreated with the LT synthesis inhibitor MK886 (1 µM) for 20 min. (B) AMs from C57BL/6 and 129/Sv mice were incubated for 2 h with IgG-opsonized or non-opsonized yeast cells at a yeast-to-cell ratio of 1∶5. (C) PMs were pretreated with the LT synthesis inhibitor MK886 (1 µM) for 20 min, followed by challenge with LTB₄ (10 nm) for 5 min before the infection (C). The data are expressed as the mean ± SEM from one representative experiment of a total of two experiments (n = 3 to 5). *sv129 compared with C57BL/6. ^#sv129 and C57BL/6 compared with MK886/LTB₄ treatment. p<0.05 was considered significant.</p

Erythropoietin Exacerbates Inflammation and Increases the Mortality of Histoplasma capsulatum-Infected Mice

Erythropoietin (EPO) is a key hormone involved in red blood cell formation, but its effects on nonerythroid cells, such as macrophages, have not been described. Macrophages are key cells in controlling histoplasmosis, a fungal infection caused by Histoplasma capsulatum (Hc). Considering that little is known about EPO’s role during fungal infections and its capacity to activate macrophages, in this study we investigated the impact of EPO pretreatment on the alveolar immune response during Hc infection. The consequence of EPO pretreatment on fungal infection was determined by evaluating animal survival, fungal burden, activation of bronchoalveolar macrophages, inflammatory mediator release, and lung inflammation. Pretreatment with EPO diminished mononuclear cell numbers, increased the recruitment of F4/80+/CD80+ and F4/80+/CD86+ cells to the bronchoalveolar space, induced higher production of IFN-γ, IL-6, MIP-1α, MCP-1, and LTB4, reduced PGE2 concentration, and did not affect fungal burden. As a consequence, we observed an increase in lung inflammation with extensive tissue damage that might account for augmented mouse mortality after infection. Our results demonstrate for the first time that EPO treatment has a deleterious impact on lung immune responses during fungal infection