Involvement of the capsular GalXM-induced IL-17 cytokine in the control of Cryptococcus neoformans infection

Abstract

Cryptococcus neoformans is an opportunistic fungus that can cause lethal brain infections in immunosuppressed individuals. Infection usually occurs via the inhalation of a spore or desiccated yeast which can then disseminate from the lung to the brain and other tissues. Dissemination and disease is largely influence by the production of copious amounts of cryptococcal polysaccharides, both which are secreted to the extracellular environment or assembled into a thick capsule surrounding the cell body. There are two important polysaccharides: glucuronoxylomannan (GXM) and galactoxylomannan, also called as glucuronoxylomanogalactan (GXMGal or GalXM). Although GXM is more abundant, GalXM has a more potent modulatory effect. In the present study, we show that GalXM is a potent activator of murine dendritic cells, and when co-cultured with T cells, induces a Th17 cytokine response. We also demonstrated that treating mice with GalXM prior to infection with C. neoformans protects from infection, and this phenomenon is dependent on IL-6 and IL-17. These findings help us understand the immune biology of capsular polysaccharides in fungal pathogenesis.

Figure 1

CD86 and MHC II expression and cytokine production in murine dendritic cells stimulated with GXM or GalXM capsular polysaccharides. Dendritic cells (2 × 10⁵/well) were incubated for 24 h in the presence of 50 μg/mL of capsular GXM or GalXM (wild-type strain B3501) from Cryptococcus neoformans or 10-ng/mL lipopolysaccharide (LPS) in the presence or absence of polymyxin B (100 ng/mL). Cells with labeled (A) CD11c (phycoerythrin; PE) and CD86 (FITC) or (B) CD11c (PE) and MHC II (FITC) were checked by flow cytometry along with positive cells. The control corresponds to dendritic cells without stimulus. The graph represents the mean fluorescence intensity (MFI). The result represents one of three independent experiments. The asterisks indicate ***P < 0.001, **P < 0.01, and *P < 0.05 compared to the control, and the hash symbol (#) indicates P < 0.01 compared to LPS without the addition of polymyxin B. To test cytokine production after stimulation, the culture supernatants were collected, and the production of cytokines IL-12p40 and IL-10 were determined with an ELISA assay. The control corresponds to dendritic cells without stimulus. The results represent one of four independent experiments. The asterisks indicate ***P < 0.001 and **P < 0.01 in relation to the control.

Figure 2

The effect of different concentrations of GXM and GalXM on the production of IL-12p40 by dendritic cells. Dendritic cells (2 × 10⁵/well) were incubated in the presence of different concentrations (μg/mL) of (A) GXM or (B) GalXM for 24 hours. The culture supernatants were then collected, and ELISA assays were used to determine the amount of the cytokine IL-12p40. The control (100 ng/mL LPS) corresponds to dendritic cells without stimulus. The results represent one of four independent experiments. The asterisks indicate ***P < 0.001 in relation to the control.

Figure 3

Increased in vitro lymphoproliferative response induced by murine dendritic cells treated with GXM or GalXM. Dendritic cells (3 × 10⁴/well) were incubated for 24 h in the presence of 50 μg/mL of the constituents’ capsule of C. neoformans. The dendritic cells were then incubated with (A) MLN cells (3 × 10⁵/well) or with (B) MLN (3 × 10⁵/well) in the presence of ConA (5 μg/mL). After 72 h, lymphoproliferation was analyzed by the incorporation of 3H-thymidine in the last 18 h of culture. Lipopolysaccharide (LPS) was used at a concentration of 100 ng/mL. (C) Dendritic cells were incubated with MLN cells (3 × 10⁵/well). After 24 or 48 hours, T cells were labeled with anti-CD4 (allophycocyanin) and the percentage of apoptotic cells was quantified by flow cytometry using annexin V (FITC) and propidium iodide. Dexamethasone (1 μM) was used as a positive control for apoptosis. The results represent one of three independent experiments. The asterisks indicate ***P < 0.001 and *P < 0.05 relative to the control.

Figure 4

CD4⁺ T cells in culture with murine dendritic cells pretreated with GalXM secrete IFN-γ. Dendritic cells (3 × 10⁴/well) were incubated for 24 h in the presence of 50 μg/mL of the constituents’ capsule of C. neoformans. The dendritic cells were then incubated with CD4⁺ T cells (3 × 10⁵/well) for 48 h. The culture supernatant was collected, and ELISA assays were used to determine the production of (A) IFN-γ and (B) IL-4. ConA was used at 5 μg/mL, and anti-IFN-γ and its IgG1a κ control were used at a concentration of 3 μg/mL. The control corresponds to dendritic cells without stimulus. The results represent one of four independent experiments. The asterisks indicate **P < 0.01 and *P < 0.05 relative to the control.

Figure 5

Murine dendritic cells pre-treated with capsular GalXM in co-culture with CD4⁺ T cells induce the production of cytokines that direct the Th17 profile. Dendritic cells (3 × 10⁴/well) were incubated for 24 h in the presence of 50 μg/mL of capsular GXM or GalXM from C. neoformans. The dendritic cells were then incubated with CD4⁺ T cells (3 × 10⁵/well) for 48 h. The culture supernatant was collected, and the cytokines (A) TGF-β, (B) IL-6, and (C) IL-17 were analyzed with ELISA assays. The αCD3 and LPS were used at 5 μg/mL and 100 ng/mL, respectively. The control corresponds to dendritic cells without stimulus. The results represent one of four independent experiments. The asterisks indicate ***P < 0.001, **P < 0.01, and *P < 0.05 relative to the control.

Figure 6

Treatment with GalXM induces the removal of fungal cells from the organs of C57BL/6 mice infected with C. neoformans. Mice were pretreated with PBS or capsular GalXM (250 μg/mL intratracheally) 24 h prior to an intratracheal injection with 10⁶ C. neoformans cells. After 14 days, the mice were euthanized and the brain, spleen, and lungs were recovered. Viable fungal cells were quantified in petri dishes containing agar from the respective homogenized organs: (A) brain, (B) spleen, and (C) lung. (D) Illustrative picture of a lung count. The results are expressed in CFUs (dilutions: 1:2 for brain and spleen; 1:100 for lung). The results represent one of three independent experiments (n = 5 mice/group). The asterisks indicate ***P < 0.001 and **P < 0.01 relative to the control.

Figure 7

The capsular GalXM constituent induces IL-6 production in bronchoalveolar lavage cells from C57BL/6 mice. Total bronchoalveolar lavage cells (1 × 10⁵/well) were incubated for 24 h before adding 50 μg/mL of capsular GalXM. The culture supernatant was collected at Days 2, 4 and 7 after adding the GalXM, and IL-6 production was determined with ELISA assays. The results represent one of two independent experiments.

Figure 8

GalXM loses the ability to induce the removal of fungal cells in the lung of C. neoformans-infected IL-6^−/− mice. Mice were pretreated with PBS or capsular GalXM (250 μg/mL intratracheally) 24 h prior to an intratracheal injection with 10⁶ C. neoformans cells. After 30 days, the mice were euthanized, and the lungs were recovered. Viable fungal cells were quantified in petri dishes containing agar from the homogenized organ. The results are expressed in CFUs (dilutions: 1:10 for wild type; 1:1000 for IL-6^−/−). The results represent one of four independent experiments (n = 5 mice/group). The asterisk indicates *P < 0.05 relative to the control. The hash symbol (#) indicates P < 0.001 compared to the wild-type mice.

Figure 9

IL-6^−/− mice infected with C. neoformans produce increased numbers of fungal cells. Interleukin-6 deficient mice (D–F) were intratracheally pretreated with PBS (B,E) or 250 μg/mL capsular GalXM (C,F) 24 h before an intratracheal injection with 10⁶ C. neoformans cells. After 30 days, the mice were euthanized, and the lungs were recovered. Fungal cells were identified in histological sections positive for mucicarmine. The black arrows in Panels B and C indicate C. neoformans. Panels E and F show an abundance of C. neoformans. The results represent one of three independent experiments (n = 5 mice/group). The photographs were taken under an optical microscope at 100× (A–C) and 200 × (D–F) magnification.

Figure 10

IL-17^−/− mice treated with GalXM present a defect similar to that seen in IL6^−/− mice in the control of pulmonary infection. Mice (Wt, IL6^−/− and IL17^−/−) were intratracheally pretreated with 250 μg/mL capsular GalXM 24 h before an intratracheal injection with 10⁶ C. neoformans cells. After 30 days, the mice were euthanized, and the lungs were recovered. Viable fungal cells were quantified in petri dishes containing agar from the homogenized organ. The results are expressed in CFUs (dilutions: 1:10 for wild type; 1:1000 for IL-6^−/− and IL17^−/−). The results represent one of four independent experiments (n = 5 mice/group). The asterisk indicates *P < 0.05 relative to the control.