Histoplasma capsulatum manifests preferential invasion of phagocytic subpopulations in murine lungs

Abstract

Numerous in vitro studies have demonstrated that Histoplasma capsulatum is engulfed by the diverse populations of phagocytic cells including monocytes/macrophages (Mphi), immature dendritic cells (DC), and neutrophils. The in vivo distribution of H. capsulatum has yet to be examined following an intrapulmonary challenge. To accomplish this goal, we engineered GFP into two genetically dissimilar strains of H. capsulatum, G217B and 186R. C57BL/6 mice were infected with each of these strains, and we analyzed the distribution of this fungus in the three major phagocytic populations on successive days. Yeast cells were found in all three populations of cells from Days 1 through 7. Proportionally, DC dominated at Day 1, whereas the majority of yeast cells was detected in neutrophils thereafter. Yeast cells were present in inflammatory and resident Mphi on Day 3, but on Day 7, they were chiefly in inflammatory Mphi. Yeast cells were predominantly in a CD11c(+intermediate/high), F4/80(-), CD11b(+), Ly-6C(+), CD205(+) DC population. Neutralization of TNF-alpha or IFN-gamma produced a significant redistribution of yeast cells. These results reveal the complex nature of intracellular residence of this fungus. Moreover, the findings demonstrate that there is a skewing in the subpopulations of cells that are infected, especially DC.

Fig. 1.

Flow cytometry profile of G217B and G186R engineered to express GFP. Yeast cells were harvested from cultures of actively growing yeast cells and analyzed by flow cytometry (B and E). Controls were cells not expressing GFP (A and D). (C and F) Representative dot-plots of GFP⁺ cells in mouse lungs from Day 7 of infection.

Fig. 2.

Analysis of Mφ, PMN, and DC from the lungs of mice infected with G217B or G186R. Mice were infected with 2 × 10⁶ yeast cells i.n., and cells were analyzed on Days 1, 3, 5, and 7 of infection. Mφ were identified as Mac-3⁺, CD11c^–/lo cells, high autofluorescence; PMN by Ly-6G⁺, Mac3⁻, and CD11c^–cells; and DC as Mac-3⁻, CD11c^{intermediate/high}, I-A^bhi, low autofluorescence. Data represent pooled mean ± sem of n = 7–9 mice in two or three separate experiments. *, P < 0.05; **, P < 0.01.

Fig. 3.

Distribution of lung phagocytes that harbor yeast cells. Mice were infected with 2 × 10⁶ yeast cells, and at Days 1, 3, 5, and 7 postinfection, lung leukocytes were stained and analyzed. Cells were gated on GFP, and a proportion of surface-positive cells was assessed. Absolute numbers were calculated by multiplying the number of leukocytes × the proportion of a positive cell population that harbored GFP⁺ yeast cells. Data represent mean ± sem of seven to nine mice/group performed in two or three separate experiments. *, P < 0.05, compared with controls; **, P < 0.01, compared with controls.

Fig. 4.

Effect of neutralization of TNF-α or IFN-γ on in vivo residence of GFP⁺ yeast cells. Mice were infected with 2 × 10⁶ yeast cells and given rat IgG, mAb to TNF-α, or IFN-γ i.p. Preliminary data indicated that there were no differences between mice receiving PBS or rat IgG. At Days 3 and 7 postinfection, lung leukocytes were analyzed for the proportion of a given population that contained GFP⁺ yeast cells. Data represent mean (±sem) of n = 6–8 mice performed in two or three individual experiments. *, P < 0.05, compared with controls; **, P < 0.01, compared with controls.

Fig. 5.

Effect of neutralization of TNF-α or IFN-γ on in vivo residence of GFP⁺ yeast cells on Mφ (A–D) and DC (E–H) subsets. Mice were infected and treated as described in Figure 4. Data were analyzed as percentage of cells that were infected. Data represent mean (±sem) of n = 5–7. *, P < 0.05, compared with controls; **, P < 0.01, compared with controls.

Fig. 6.

Representative dot-plots of phagocytes from mice infected with G217B at Days 3 and 7. Cells were gated on GFP⁺ cells and analyzed for expression of Mac-3 and Ly-6C (top panels), Mac-3 and CD62L (middle panels), and CD11c and CD205 (bottom panels).

Fig. 7.

Effect of neutralization of TNF-α or IFN-γ on in vivo residence of GFP⁺ yeast cells in Mφ (A–D) and DC (E–H) subsets. Mice were infected and treated as described in Figure 4. Absolute numbers of infected cells were calculated. Data represent mean (±sem) of n = 5–7 in two separate experiments. *, P < 0.05, compared with controls; **, P < 0.01, compared with controls.

Fig. 8.

Antagonism of TNF-α or IFN-γ alters the number of infected DC expressing CD80, CD86, or class II MHC (I-A^b). Mice were infected with 2 × 10⁶ yeast cells i.n. and given rat IgG, mAb to TNF-α, or IFN-γ. Cells were stained with CD11c and gated on the GFP⁺ population. Data represent mean (±sem) of n = 5–7 performed in two separate experiments. *, P < 0.05; **, P < 0.01.