5-Lipoxygenase deficiency impairs innate and adaptive immune responses during fungal infection - PubMed (original) (raw)

5-Lipoxygenase deficiency impairs innate and adaptive immune responses during fungal infection

Adriana Secatto et al. PLoS One. 2012.

Abstract

5-Lipoxygenase-derived products have been implicated in both the inhibition and promotion of chronic infection. Here, we sought to investigate the roles of endogenous 5-lipoxygenase products and exogenous leukotrienes during Histoplasma capsulatum infection in vivo and in vitro. 5-LO deficiency led to increased lung CFU, decreased nitric oxide production and a deficient primary immune response during active fungal infection. Moreover, H. capsulatum-infected 5-LO(-/-) mice showed an intense influx of neutrophils and an impaired ability to generate and recruit effector T cells to the lung. The fungal susceptibility of 5-LO(-/-) mice correlated with a lower rate of macrophage ingestion of IgG-H. capsulatum relative to WT macrophages. Conversely, exogenous LTB4 and LTC4 restored macrophage phagocytosis in 5-LO deficient mice. Our results demonstrate that leukotrienes are required to control chronic fungal infection by amplifying both the innate and adaptive immune response during histoplasmosis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. LTB4 and CysLTs production in lung tissue.

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 H. capsulatum. Data are presented as the mean ± SEM and are representative of one of two independent experiments (n = 6). *, p<0.05 vs. PBS.

Figure 2. Effect of 5-LO deficiency on survival, fungal burden and NO2 − production.

(A) 5-LO−/− and WT mice were infected i.t. with 3×106 yeast H. capsulatum 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 H. capsulatum 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.

Figure 3. 5-LO deficiency increases the inflammatory response in the lung.

Representative lung sections from WT and 5-LO−/− mice infected with H. capsulatum (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 Material and Methods section from mice after i.t. injection of PBS or i.t. infection with H. capsulatum. 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.

Figure 4. 5-LO deficiency affects the ability of macrophages to phagocytose H. capsulatum.

(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 H. capsulatum. Phagocytosis was calculated as described in the Material and Methods 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.

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

Cells were obtained as described in the Material and Methods section from mice after i.t. injection of PBS or i.t. infection with H. capsulatum. The lymphocyte population was gated for forward/side scatters and analyzed the percentage of T cells expressing a phenotype effector (CD44high/CD62low). (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.

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5-Lipoxygenase deficiency impairs innate and adaptive immune responses during fungal infection - PubMed (original) (raw)