Neuraminidase and SIGLEC15 modulate the host defense against pulmonary aspergillosis

Abstract

Influenza-associated pulmonary aspergillosis (IAPA) has been reported increasingly since the advent of use of neuraminidase (NA) inhibitors following the 2009 influenza pandemic. We hypothesize that blocking host NA modulates the immune response against Aspergillus fumigatus. We demonstrate that NA influences the host response against A. fumigatus in vitro and that oseltamivir increases the susceptibility of mice to pulmonary aspergillosis. Oseltamivir impairs the mouse splenocyte and human peripheral blood mononuclear cell (PBMC) killing capacity of A. fumigatus, and adding NA restores this defect in PBMCs. Furthermore, the sialic acid-binding receptor SIGLEC15 is upregulated in PBMCs stimulated with A. fumigatus. Silencing of SIGLEC15 decrease PBMC killing of A. fumigatus. We provide evidence that host NA activity and sialic acid recognition are important for anti-Aspergillus defense. NA inhibitors might predispose individuals with severe influenza to invasive aspergillosis. These data shed light on the pathogenesis of invasive fungal infections and may identify potential therapeutic targets.

Keywords: SIGLEC15; aspergillosis; neuraminidase; oseltamivir.

Graphical abstract

Figure 1

NA modulates the host response of PBMCs against A. fumigatus (A) TNF-α and IL-1β concentrations in culture supernatants of PBMCs (n = 12) stimulated with 1 × 10⁷/mL of mock- or NA-treated A. fumigatus conidia. (B and C) PBMCs or neutrophils (n = 12) were stimulated with A. fumigatus conidia in the presence or absence of NA, and concentrations of TNF-α, IL-1β, or IL-8 were measured from 24-h-cultured supernatants. (D) Gene expression profiling of the NEU genes in PBMCs (n = 8) that were stimulated with heat-killed A. fumigatus conidia for 4 and 24 h. Data are presented as mean ± SEM. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

Figure 2

Oseltamivir affects the susceptibility of immunocompetent mice to A. fumigatus infection (A) Immunocompetent C57BL/6J mice were treated with PBS (n = 20) or oseltamivir (10 mg/kg) (n = 20) twice daily for 5 consecutive days. On day 3 of treatment, mice were infected intranasally with A. fumigatus or remained uninfected (naive). All mice were sacrificed on days 1 and 3 after infection. (B) Fungal burden in lung homogenates on days 1 and 3 after infection (log10). (C) Production of TNF-α, IL-1β, IL-6, and IL-10 was measured from lung homogenates of uninfected (striped bars) or infected (empty bars) mice that were treated with oseltamivir (red bars) or remained untreated (black bars). (D) The total number of immune cells in lung homogenates from infected or uninfected mice treated with oseltamivir or controls. (E) Representative histology of the lung tissue sections of the mice on day 1 and day 3 after infection, stained with H&E. Data are presented as mean ± SEM. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

Figure 3

The effect of oseltamivir on immunocompromised mice (A and B) Corticosteroid-treated C57BL/6J mice were treated with PBS or oseltamivir (10 mg/kg) twice daily for 5 days and infected with A. fumigatus (1 × 10⁷) via the intranasal route. (A) Kaplan-Meier survival plot of mice infected with A. fumigatus. Results were derived from three independent experiments (total n = 24 mice per group). (B) Fungal burden in lung homogenates of mice on day 2 after infection. ∗∗∗p < 0.001. (C and D) BALB/c mice were treated with oseltamivir (10 mg/kg, n = 10) or PBS (n = 10) twice per day for 5 days and infected intranasally with A. fumigatus. Immunosuppression was induced by intraperitoneal administration of cyclophosphamide (150 mg/kg). Shown are Kaplan-Meier survival plot (C) from mice after 3 days of infection and fungal burden (D) measured from lung homogenates.

Figure 4

Oseltamivir treatment impaired the fungal killing capacity of mouse splenocytes and human PBMCs (A) Killing capacity of splenocytes from immunocompetent C57BL/6J mice treated with (red bars) or without (black bars) oseltamivir and infected with A. fumigatus (1 × 10⁶/mL) (empty bars) or left uninfected (striped bars). (B and C) PBMCs and neutrophils were stimulated with A. fumigatus conidia (MOI of 4:1) in the presence or absence of oseltamivir carboxylate (n = 16). A killing assay was performed after 4 h of incubation at 37°C. Cells were lysed with H₂O and plated on Sabouraud dextrose agar (SDA) in serial dilutions, and the remaining CFUs were counted after 24-h incubation at 37°C. (D and E) ROS production from immune cells in lung homogenates of immunocompetent mice treated with or without oseltamivir and infected with A. fumigatus or left uninfected. (F and G) ROS induction from PBMCs and neutrophils stimulated with A. fumigatus conidia in the presence or absence of oseltamivir carboxylate (n = 11). Data are shown as mean ± SEM. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

Figure 5

The role of SIGLEC15 in the host response against A. fumigatus (A and B) PBMCs or neutrophils were stimulated with A. fumigatus for 4 h in the presence or absence of exogenous C. perfringens NA (n = 14). Cells were then lysed in H₂O, and the remaining conidia were plated in serial dilutions on SDA and incubated for 24 h at 37°C. (C) Gene expression profiling of SIGLEC genes, using Illumina Human HT-12 Expression BeadChip, in PBMCs stimulated with heat-killed A. fumigatus conidia for 4 and 24 h (n = 8). (D) Recombinant SIGLEC15 (recSIGLEC15) binding to A. fumigatus. Green, heat-killed A. fumigatus; blue, heat-killed A. fumigatus + secondary antibody (anti immunoglobulin G [IgG]-Alexa 647); red, heat-killed A. fumigatus + recSIGLEC15 + secondary antibody. (E) PBMCs were transfected with SIGLEC15 siRNA or scrambled control siRNA for 24 h prior to stimulation with A. fumigatus conidia. After 4 h of stimulation, cells were lysed with H₂O, and a killing assay was performed as described previously (n = 12). Data are represented as mean ± SEM (∗p < 0.05). (F) Killing capacity of PBMCs transfected with SIGLEC15 siRNA or control siRNA, as described previously, and stimulated with NA-treated or mock-treated A. fumigatus conidia (n = 6). (G) A. fumigatus-induced cytokine production from PBMCs of individuals harboring a polymorphism in the SIGLEC15 gene (rs2919643) (TT = 12, CT/CC = 7). Data are represented as mean ± SEM (∗∗p < 0.01, ∗p < 0.05).