The Major Surface Glycoprotein of Pneumocystis murina Does Not Activate Dendritic Cells

Abstract

The major surface glycoprotein (Msg) is the most abundant surface protein among Pneumocystis species. Given that Msg is present on both the cyst and trophic forms of Pneumocystis and that dendritic cells play a critical role in initiating host immune responses, we undertook studies to examine activation of bone marrow-derived myeloid dendritic cells by Msg purified from Pneumocystis murina. Incubation of dendritic cells with Msg did not lead to increased expression of CD40, CD80, CD86, or major histocompatibility complex class II or to increased secretion of any of 10 cytokines. Microarray analysis identified very few differentially expressed genes. In contrast, lipopolysaccharide-activated dendritic cells had positive results of all of these assays. However, Msg did bind to mouse mannose macrophage receptor and human DC-SIGN, 2 C-type lectins expressed by dendritic cells that are important in recognition of pathogen-associated high-mannose glycoproteins. Deglycosylation of Msg demonstrated that this binding was dependent on glycosylation. These studies suggest that Pneumocystis has developed a mechanism to avoid activation of dendritic cells, potentially by the previously identified loss of genes that are responsible for the high level of protein mannosylation found in other fungi.

Figure 1.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified Pneumocystis murina major surface glycoprotein (Msg). Msg was purified by either SDS extraction or lyticase treatment followed by affinity purification using a concanavalin A column, as described in the methods, after which the samples were analyzed by SDS-PAGE and staining with Coomassie blue. Lane 1, SDS-Msg; lane 2, lyticase-Msg. Lyticase treatment leads to a small decrease in molecular weight of the major band, as previously reported [15].

Figure 2.

Flow cytometry of dendritic cell activation by lipopolysaccharide (LPS) and major surface glycoprotein (Msg). Immature mouse dendritic cells were generated in vitro, using granulocyte-macrophage colony-stimulating factor, and were either unstimulated or stimulated with LPS (300 ng/mL), sodium dodecyl sulfate (SDS)–Msg (5 µg/mL), or lyticase-Msg (5 µg/mL), for 24 hours. Dendritic cells were identified on the basis of CD11c expression, and the surface expression of CD80, CD86, CD40, and major histocompatibility complex (MHC) class II were measured by flow cytometry. The expression of activation markers was analyzed by FlowJo and compared to expression for the untreated-dendritic cells. The light gray line represents untreated dendritic cells, and the black line represents the stimulated population. The x-axis is fluorescence intensity, and the y-axis is normalized to mode, using FlowJo software. LPS (top row) led to increased expression of activation markers, while neither Msg preparation led to increased expression of any activation marker. Results are for 1 representative experiment of 4 experiments, all with similar results.

Figure 3.

Cytokine production by dendritic cells following activation. Positively selected (CD11c⁺) immature dendritic cells (10⁶ cells/well) were either unstimulated or stimulated with lipopolysaccharide (LPS; 300 ng/mL), crude Pneumocystis murina antigen (20 µg/mL), β-glucan (300 ng/mL), sodium dodecyl sulfate (SDS)–major surface glycoprotein (Msg; 5 µg/mL), or lyticase-Msg (5 µg/mL) for 48 hours. Supernatants were collected, and levels of 10 cytokines were analyzed as described in Methods. Significant increases were seen for multiple cytokines following stimulation with LPS and, to a lesser extent, with β-glucan but not with either of the Msg preparations. Graphed values are the average ± SD of 3 independent experiments. The ratio paired t test was used to compare each stimulated sample with the unstimulated control. *P < .05 and **P < .01. IL-1β, interleukin 1β; IL-2, interleukin 2; IL-4, interleukin 4; IL-5, interleukin 5; IL-6, interleukin 6; IL-10, interleukin 10; IL-12p70, interleukin 12p70; TNF-α, tumor necrosis factor α.

Figure 4.

Enzyme-linked immunosorbent assay (ELISA) evaluating CLR binding to major surface glycoprotein (Msg). Attachment of concanavalin A (ConA), mouse macrophage mannose receptor (MMR) or human DC-SIGN to Pneumocystis murina or Pneumocystis carinii Msg was evaluated by an ELISA format as described in Methods. Wells were coated with P. murina (top) or P. carinii (bottom) SDS-Msg (1 µg/mL of each) and, after blocking, were incubated with concanavalin A (ConA; 2 µg/mL), MMR (10 µg/mL), or DC-SIGN (10 µg/mL). For P. murina, inhibition with mannan (100 µg/mL) was evaluated in parallel wells. All 3 lectins bound to both Msgs, although the binding of MMR to P. murina was of borderline statistical significance (P = .053). Mannan significantly inhibited binding of both MMR and DC-SIGN to P. murina Msg. Results represent the mean of 2–4 individual experiments for each condition. The ratio paired t test was used to compare lectin binding to control (no lectin) and to compare samples incubated with mannan plus lectin to those incubated with lectin alone. *P < .05, **P < .01, and ***P < .001, for comparison of lectin to control; and ****P < .05, for comparison of lectin alone to lectin plus mannan.

Figure 5.

Immunoblot and deglycosylation studies of C-type lectin receptor binding to major surface glycoprotein (Msg). Pneumocystis murina sodium dodecyl sulfate (SDS)–Msg and lyticase-Msg were deglycosylated as described in Methods, and untreated or deglycosylated preparations were separated by SDS polyacrylamide gel electrophoresis and stained with Coomassie blue (left) or transferred to a nitrocellulose membrane and incubated with mouse macrophage mannose receptor (MMR) or DC-SIGN followed by detection reagents (right). As seen in the left panel, deglycosylation leads to a small decrease in the apparent molecular weight of both Msg bands (indicated by the bracket). As seen in the right panel, both MMR and DC-SIGN bind to untreated Msg but no longer bind following deglycosylation.