Type I IFN Induction via Poly-ICLC Protects Mice against Cryptococcosis

Abstract

Cryptococcus neoformans is the most common cause of fungal meningoencephalitis in AIDS patients. Depletion of CD4 cells, such as occurs during advanced AIDS, is known to be a critical risk factor for developing cryptococcosis. However, the role of HIV-induced innate inflammation in susceptibility to cryptococcosis has not been evaluated. Thus, we sought to determine the role of Type I IFN induction in host defense against cryptococci by treatment of C. neoformans (H99) infected mice with poly-ICLC (pICLC), a dsRNA virus mimic. Unexpectedly, pICLC treatment greatly extended survival of infected mice and reduced fungal burdens in the brain. Protection from cryptococcosis by pICLC-induced Type I IFN was mediated by MDA5 rather than TLR3. PICLC treatment induced a large, rapid and sustained influx of neutrophils and Ly6Chigh monocytes into the lung while suppressing the development of eosinophilia. The pICLC-mediated protection against H99 was CD4 T cell dependent and analysis of CD4 T cell polyfunctionality showed a reduction in IL-5 producing CD4 T cells, marginal increases in Th1 cells and dramatic increases in RORγt+ Th17 cells in pICLC treated mice. Moreover, the protective effect of pICLC against H99 was diminished in IFNγ KO mice and by IL-17A neutralization with blocking mAbs. Furthermore, pICLC treatment also significantly extended survival of C. gattii infected mice with reduced fungal loads in the lungs. These data demonstrate that induction of type I IFN dramatically improves host resistance against the etiologic agents of cryptococcosis by beneficial alterations in both innate and adaptive immune responses.

Fig 1. Treatment of mice with pICLC protects against C. neoformans infection through the MDA5-dependent induction of type I interferon.

Mice were intrapharyngeally infected with 5000 CFU of C. neoformans H99 and treated with polyICLC twice weekly starting on the day of infection or left untreated. Survival and fungal burdens on day 28 post-infection in WT and Ifnar1 ^-/- (A), Tlr3 ^-/- (B), and Mda5 ^-/- mice (C). IFNα (D) and IFNβ (E) concentrations in lung homogenate were measured by ELISA on day 28 post-infection. Cytokine concentrations were normalized to the total content of protein in the sample. (F) Survival of treated or untreated Ifnb ^-/- infected mice. (G) WT mice were infected and either given no treatment or treated with pICLC or rIFNα and monitored for survival. Data are representative of at least 2 independent experiments with n = 5–6 mice/group. P values for comparisons of fungal burdens are calculated based on log₁₀ transformed values.

Fig 2. Treatment of mice with pICLC synergizes with FLC to protect against C. neoformans.

Mice were intrapharyngeally infected with 5000 CFU of C. neoformans H99 and left untreated, intrapharyngeally administered pICLC, twice weekly, treated with fluconazole via daily intraperitoneal injections, or given both treatments concomitantly. Treatments were started 24 h after infection. Brains and lungs were harvested on days 7, 14, and 28 to measure fungal burdens. Data are representative of 3 experiments with n = 3–5 mice/group.

Fig 3. Delivery of pICLC into the airways induces the rapid influx of neutrophils and Ly6C^high monocytes into the lung tissue parenchyma and suppresses C. neoformans induced eosinophilia.

(A) Gating strategy used in these experiments to identify distinct myeloid cell subsets. Example plots are taken from the lungs of a naïve animal. (B-D) Naïve and H99 infected mice were intrapharyngeally administered PBS or pICLC once at time of infection and myeloid cell responses were analyzed on day 3. To discriminate intravascular and parenchymal leukocytes, mice were intravenously injected with anti-CD45.2-FITC and euthanized 3 minutes later. (B) Example FACS plots of the intravascular stain after gating on Ly6C^high monocytes as shown in (A). Percentage (C) and absolute number (D) of each myeloid cell type that is intravascular stain negative in the lung. Data in (B-D) on day 3 post-infection are pooled from 2 separate experiments with n = 5 mice/group. (E) The percentage of total live lung cells that are each myeloid cell type in untreated and pICLC treated mice on day 20 post-infection. (F) Number of each myeloid cell type in the lungs that is intravascular stain negative on day 20 post-infection. Data were pooled from two independent experiments with n = 4–5 mice/group.

Fig 4. PICLC treatments enhances Th17 responses in C. neoformans infected mice.

I-A ^b-/- mice were intrapharyngeally infected with 5000 CFU of H99 and either left untreated or given pICLC and monitored for survival (A) or sacrificed on day 27 to measure fungal burdens in the lungs and brains (B). Data are representative of three independent experiments with n = 5 mice/group. (C) RORγt and GATA-3 intracellular staining in CD44^highfoxp3^- effector CD4 T cells isolated from the lungs of treated and untreated mice on day 20 post-infection. Example FACS plots (D) and summary data (E) of intracellular cytokine production by lung CD44^highfoxp3^- effector CD4 T cells following restimulation with anti-CD3/anti-CD28 on day 28 post-infection. Data are pooled from 2 separate experiments with n = 5 mice/group. (F) Cytokine polyfunctionality of effector CD4 T cells in the lungs of untreated or pICLC treated mice on day 28 post-infection. Data are pooled from 2 separate experiments with n = 5 mice/group. P values for comparisons of fungal burdens are calculated based on log₁₀ transformed values.

Fig 5. IL-17A and IFNγ contribute to pICLC induced protection against C. neoformans infection.

(A) WT mice were intrapharyngeally infected with 5000 CFU of H99 and either left untreated, given pICLC, anti-IL-17A, or both pICLC and anti-IL-17A concomitantly. Lungs were harvested on day 20 and fungal burdens were quantified. Results from 2 independent experiments are shown separately. Ifng ^-/- mice were intrapharyngeally infected with 5000 CFU of H99 and either left untreated or given pICLC and monitored for survival (B) or sacrificed on d28 to quantify fungal burdens in the lungs and brains (C). Data are representative of 3 independent experiments with n = 3–5 mice/group. P values for comparisons of fungal burdens are calculated based on log₁₀ transformed values.

Fig 6. PICLC treatment protects against pulmonary infection with C. gattii.

WT mice were intrapharyngeally infected with 5000 CFU of C. gattii R265 and either left untreated or given pICLC and monitored for survival (A) or sacrificed on day 28 to measure fungal burdens in the lungs and brains (B). Data are representative of 3 experiments with n = 5–6 mice per group. P values for comparisons of fungal burdens are calculated based on log₁₀ transformed values.