Glycosphingolipids mediate pneumocystis cell wall β-glucan activation of the IL-23/IL-17 axis in human dendritic cells

Abstract

Pneumocystis species are opportunistic fungal organisms that cause severe pneumonia in immune-compromised hosts, with resultant high morbidity and mortality. Recent work indicates that IL-17 responses are important components of host defense against fungal pathogens. In the present study, we demonstrate that cell-surface β-glucan components of Pneumocystis (PCBG) stimulate human dendritic cells (DCs) to secrete IL-23 and IL-6. These cytokines are well established to stimulate a T helper-17 (Th17) phenotype. Accordingly, we further observe that PCBG-stimulated human DCs interact with lymphocytes to drive the secretion of IL-17 and IL-22, both Th17-produced cytokines. The activation of DCs was shown to involve the dectin-1 receptor with a downstream activation of the Syk kinase and subsequent translocation of both the canonical and noncanonical components of the NF-κB transcription factor family. Finally, we demonstrate that glycosphingolipid-rich microdomains of the plasma membrane participate in the activation of DCs by PCBG through the accumulation of lactosylceramide at the cell surface during stimulation with PCBG. These data strongly support the idea that the β-glucan surface components of Pneumocystis drive the activation of the IL-23/IL-17 axis during this infection, through a glycosphingolipid-initiated mechanism.

Figure 1.

Activation by β-glucan components of Pneumocystis (PCBG) of the IL-23/IL-17 axis. (A) IL-23, (B) IL-6, and (C) transforming growth factor (TGF)–β were measured by ELISA in the supernatant of PCBG-stimulated or unstimulated (NT) monocyte-derived dendritic cells (DCs). (D) As a control for specificity, IL-23 production by DCs was also measured after stimulation with the soluble β-glucan pustulan or with human TNF-α. For these experiments, PCBG served as positive control. (E and F) Next, particulate PCBG-activated and nonactivated DCs were cocultured in a mixed lymphocyte reaction with naive CD4 T cells for 72 hours, to measure subsequent PCBG-driven lymphocyte activation. The lymphocyte-derived cytokines, IL-17 (E) and IL-22 (F), were measured in the media supernatants of cell cultures, using ELISA. The data were analyzed using one-way ANOVA and posttest Bonferroni comparisons. Shown are data from a representative experimental run, typical of at least three separate experimental preparations, performed with different DC preparations. *P = 0.01–0.05. **P = 0.001–0.01. ***P < 0.001. ns, not significant (P > 0.05).

Figure 2.

Dectin-mediated IL-23 secretion by DCs. Human DCs were incubated for 1 hour with different concentrations of either anti–dectin-1 antibody (ab) or nonimmune IgG control, before stimulation with PCBG. IL-23 was measured using ELISA in the media supernatants of cells 18 hours after stimulation. The data were analyzed with one-way ANOVA and posttest Bonferroni comparisons. Shown are data from a representative experimental run, typical of at least three separate experimental preparations, conducted with different DC preparations. **P = 0.001–0.01. ***P < 0.001.

Figure 3.

Syk-mediated activation of dendritic cell IL-23 pathway by PCBG. (A) Immunoblot analysis of phospho-Syk (p-Syk) and total Syk in unstimulated and PCBG-stimulated human DCs at indicated culture times. Total actin was used as loading control. (B and C) IL-23 and IL-1β concentrations from unstimulated or PCBG-stimulated DCs. The Syk inhibitor piceatannol was used before stimulation with PCBG for some of the conditions, as indicated. Data were analyzed with one-way ANOVA and posttest Bonferroni comparisons. Shown are data from a representative experimental run, typical of at least three separate experimental preparations, performed with different DC preparations. *P = 0.01–0.05. ***P < 0.001.

Figure 4.

PCBG activation of canonical and noncanonical NF-κB pathways in DCs leading to IL-23 secretion. (A) DNA binding of different NF-κB subunits (p65, p-50, C-Rel, RelB, and p-52) in the nuclear extracts of unstimulated and PCBG-stimulated DCs for 1 hour and 24 hours. RelB, the transcription factor protein encoded by the human RELB gene. C-Rel, the proto-oncogene protein encoded by the human REL gene. Data are expressed as optical density (OD) at 450 nm. Shown are data from a representative experimental run, typical of at least three separate experimental preparations, performed with different DC preparations. (B) The NF-κB inhibitor BAY-11-7085 was used before and throughout stimulation with PCBG, and IL-23 concentrations were measured in media supernatants by ELISA. *P = 0.01–0.05. **P = 0.001–0.01. ***P < 0.001.

Figure 5.

Glycosphingolipids are essential for optimal IL-23/IL-17 signaling. (A) Scheme of the sphingolipid biosynthesis pathway in mammalian cells from palmitoyl+coenzyme+A and L-serine. Glycosphingolipid synthesis inhibition by PDMP (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol · HCl) and EDOP4 is outlined. (B) IL-23 secretion from PCBG-stimulated DCs in the presence of PDMP or (C) EDOP4. IL-23 was measured by ELISA 18 hours after stimulation in media supernatants of the cells. (D) Cells stimulated as already described were cocultured with CD4 T cells for 72 hours, and IL-17 was measured in the media supernatants of cultures. Shown are data from a representative experimental run, typical of at least three separate experimental preparations, performed with different DC preparations. **P = 0.001–0.01. ***P < 0.001.

Figure 6.

PCBG induces changes in the distribution of BODIPY–lactosylceramide [boron dipyrromethene (4,4-diXuoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene)] (BODIPY-LacCer) at the plasma membrane. (A) Distribution of BODIPY-LacCer at the plasma membrane after stimulation of DCs with PCBG. A group of DCs was preincubated with 20 μM of PDMP, as already indicated. Images were acquired simultaneously at both green and red wavelengths (see Materials and Methods). The dual acquisition of images permits the identification of microdomains enriched in lactosylceramide (seen as red), as distinguished from regions with lower concentrations of lactosylceramide (seen as green). (B) Red/green (R/G) fluorescent ratios were measured. The number of cells examined is shown as “n.” Representative images from at least three independent experiments are displayed. ***P < 0.001.

Figure 7.

The glycosphingolipid inhibitor PDMP alters dectin-1/Syk signaling, and alters NF-κB binding. (A) Dectin-1 was immunoprecipitated from PCBG-stimulated DCs that were treated with or without PDMP (20 μM). Unstimulated cells were used as a control samples. Phosphotyrosine antibody was used to detect dectin-1 phosphorylation in the immunoprecipitates. Total dectin-1 expression was used as a control. (B and C) Immunoblot analyses of p-Syk and phospho-extracellular regulated kinase (p-ERK; p44–42) are shown after pretreatment of DCs with PDMP or piceatannol. All inhibitors were added 1 hour before and throughout subsequent stimulation. DCs were stimulated for 15 minutes with PCBG. Tubulin was used as loading control. The data shown are representative of at least three independent experiments. (D) DNA binding of the various NF-κB subunits in the nuclear extracts of unstimulated and PCBG-stimulated cells for 24 hours. Again, the PDMP was added 1 hour before and throughout the subsequent stimulation, as indicated in the experimental conditions. The data are expressed as the fold-increase, and represent the integrated results from three independent experiments. *P = 0.01–0.05. **P = 0.001–0.01. ns, not significant (P > 0.05).