Early NK cell-derived IFN-{gamma} is essential to host defense in neutropenic invasive aspergillosis

Abstract

Invasive aspergillosis is among the most common human fungal infections and occurs in patients with severe and complex defects in immune responses. NK cells have previously been found to be important in host defense against this infection, but the mechanism of this effect is not known. We hypothesized that NK cells mediate their protective effect in invasive aspergillosis by acting as the major source of IFN-gamma during early infection. We found that, in the lungs of neutropenic mice with invasive aspergillosis, NK cells were the major population of cells capable of generating IFN-gamma during early infection. Depletion of NK cells resulted in reduced lung IFN-gamma levels and increased lung fungal load that was independent of T and B cell subsets. Depletion of NK cells and absence of IFN-gamma resulted in a similar increase in susceptibility to the infection, but depletion of NK cells in IFN-gamma-deficient hosts did not result in further increase in severity of the infection. NK cell-derived IFN-gamma caused enhanced macrophage antimicrobial effects in vitro and also resulted in greater expression of IFN-inducible chemokines in the lungs. Finally, transfer of activated NK cells from wild-type, but not IFN-gamma-deficient hosts, resulted in greater pathogen clearance from the lungs of both IFN-gamma-deficient and wild-type recipients. Taken together, these data indicate that NK cells are the main source of early IFN-gamma in the lungs in neutropenic invasive aspergillosis, and this is an important mechanism in the defense against this infection.

Figure 1

Cellular source of lung IFN-γ in neutropenic mice with invasive aspergillosis. A–B: Gating scheme and summary data of flow cytometry of lung single cell suspensions on day 1 of infection. Data are representative of 4 mice per group. C: Lung IFN-γ protein level in lung homogenates at various time points after onset of infection as measured by ELISA. Both groups were neutrophil-depleted; NK cell depletion was achieved with anti-NK1.1. Day 0 represents uninfected animals. n=10–12 mice/group in 2 separate experiments. *, p<0.05 compared to other groups.

Figure 2

Effect of NK cell depletion on lung IFN-γ and fungal content in wildtype and RAG-1 deficient mice. Lung IFN-γ levels (panel A) and chitin content (panel B) were measured on days 1 and 3 of infection, respectively. All groups were neutrophil depleted; NK cell depletion was achieved with anti-asialo-GM1. n=6 mice/group; *, p<0.05 compared to mice of the same genetic background without NK depletion.

Figure 3

Role of NK cell-derived IFN-γ in outcome of neutropenic invasive aspergillosis. A: Survival study comparing neutropenic wildtype and IFN-γ-deficient mice with invasive aspergillosis with or without NK cell depletion after infection with an LD₂₀ inoculum for neutropenic wildtype mice. n=24–35 mice/group, pooled from 3 experiments. B: Lung chitin content on day 3 on infection. n=14–18 mice/group, pooled from 3 experiments. *, p<0.05 compared to each of the other experimental groups.

Figure 4

Effect of NK cell-derived IFN-γ on macrophage antimicrobial functions in vitro. A–B: Effect of NK cells on alveolar macrophage phagocytosis and killing of Aspergillus elements. Macrophages with or without NK cells from various donors were co-cultured for 3 hours prior to addition of conidia. Phagocytosis was determined after 1 hour and fungal growth was measured after 16 hours. *, p<0.05 compared to each of the other experimental groups. C: Effect of NK cells on inflammatory macrophage killing of Aspergillus hyphae. Macrophages from wildtype mice and activated NK cells from various donors were incubated at the indicated ratios with pre-formed hyphae. *, p<0.05 compared to macrophages alone and macrophages incubated with IFN-γ-deficient NK cells.

Figure 5

Protein levels of CXCL9, CXCL10 and CXCL11 in lung homogenates in neutropenic mice with invasive aspergillosis. A: Lung chemokine levels at various time points after inoculation. Day 0 represents uninfected animals. Data represent mean ± SEM; n = 5–6 mice per time point; *, p<0.05 for increase over time for each chemokine. B: Lung chemokine levels in neutropenic C57BL/6 and IFN-γ gene knockout mice on C57BL/6 background with or without NK depletion on day 3 of infection. Data represent mean ± SEM; n = 5–6 mice per group; *, p<0.05 compared to no NK depletion; **, p<0.05 compared to wildtypes.

Figure 6

Effect of NK cell transfer on lung fungal clearance in neutropenic invasive aspergillosis. Lung chitin content on day 3 of invasive aspergillosis after infection with an LD₂₀ (panel A) or LD₁₀₀ (panel B) inocula for neutropenic mice after adoptive transfer of activated NK cells. Animals were given PBS, or activated NK cells from various donors on days 1 and 2 of infection. *, p<0.05 compared to each of the other groups. Data shown represent mean ± SEM of n=5–12 mice per group.