5-LO deficiency increases the inflammatory response in the lung.

<p>Representative lung sections from WT and 5-LO^−/− mice infected with <i>H. capsulatum</i> (A). Hematoxylin-eosin staining for leukocytes (magnifications ×100) and GMS staining for yeast cells (black arrow) (magnifications ×400). (B) Neutrophils recruitment from lung parenchyma (C) TNF-α production from homogenized lungs. Cells and cytokines were obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031701#s3" target="_blank"><i>Material and Methods</i></a> section from mice after i.t. injection of PBS or i.t. infection with <i>H. capsulatum</i>. Cells were enumerated and identified after Rosenfeld staining, and TNF-α levels were determined by ELISA. Data are expressed as the mean ± SEM from one experiment representative of a total of two experiments (n = 6). *, p<0.05 vs. PBS; #, p<0.05 vs. WT.</p

5-LO deficiency affects the ability of macrophages to phagocytose <i>H. capsulatum</i>.

<p>(A) PMs from WT and 5-LO^−/− mice were incubated for 1 h with a yeast:macrophage ratio of 1∶5 in the absence or presence of IgG. PMs were pretreated with LTB₄ (B) and LTC₄ (C) for 5 min before the addition of opsonized <i>H. capsulatum</i>. Phagocytosis was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031701#s3" target="_blank"><i>Material and Methods</i></a> section and was expressed as a percentage of the control. Data are expressed as the mean ± SEM from one experiment representative of a total of three experiments (n = 6). *, p<0.05 vs. control; #, p<0.05 vs. WT cells; & p<0.05 vs. 5-LO^−/− cells.</p

Effect of 5-LO deficiency on survival, fungal burden and NO₂⁻ production.

<p>(A) 5-LO^−/− and WT mice were infected i.t. with 3×10⁶ yeast <i>H. capsulatum</i> and survival was monitored for 30 days (n = 6). CFU numbers in lungs (B) and spleen (C) were evaluated at 7 and 14 days post <i>H. capsulatum</i> infection. (D) NO₂⁻ levels were quantified in the supernatant of lung homogenates at different time points using a Griess reaction. Data are expressed as the mean ± SEM from one experiment representative of a total of two experiments (n = 6). *, p<0.05 vs. PBS; #, p<0.05 vs. WT.</p

Deficiency of 5-LO impairs T cell recruitment to the lung.

<p>Cells were obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031701#s3" target="_blank"><i>Material and Methods</i></a> section from mice after i.t. injection of PBS or i.t. infection with <i>H. capsulatum</i>. The lymphocyte population was gated for forward/side scatters and analyzed the percentage of T cells expressing a phenotype effector (CD44^high/CD62^low). (A) CD4⁺ T cells, CD8⁺ T cells (B) and T cell proliferation(C). Data are presented as the mean ± the SEM from three experiments. *, WT and 5-LO^−/− vs. PBS; #, WT vs. 5-LO^−/−. p<0.05.</p

LTB₄ and CysLTs production in lung tissue.

<p>Enzyme immunoassay quantification of LTB₄ and CysLTs concentrations in lungs from mice that had received either an i.t. PBS injection (uninfected) or an i.t. infection with <i>H. capsulatum</i>. Data are presented as the mean ± SEM and are representative of one of two independent experiments (n = 6). *, p<0.05 vs. PBS.</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

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

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

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

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