The phagocytic capacity and immunological potency of human dendritic cells is improved by α2,6-sialic acid deficiency

Abstract

Dendritic cells (DCs) play an essential role in immunity against bacteria by phagocytosis and by eliciting adaptive immune responses. Previously, we demonstrated that human monocyte-derived DCs (MDDCs) express a high content of cell surface α2,6-sialylated glycans. However, the relative role of these sialylated structures in phagocytosis of bacteria has not been reported. Here, we show that treatment with a sialidase significantly improved the capacity of both immature and mature MDDCs to phagocytose Escherichia coli. Desialylated MDDCs had a significantly more mature phenotype, with higher expression of MHC molecules and interleukin (IL)-12, tumour necrosis factor-α, IL-6 and IL-10 cytokines, and nuclear factor-κB activation. T lymphocytes primed by desialylated MDDCs expressed more interferon-γ when compared with priming by sialylated MDDCs. Improved phagocytosis required E. coli sialic acids, indicating a mechanism of host-pathogen interaction dependent on sialic acid moieties. The DCs harvested from mice deficient in the ST6Gal.1 sialyltransferase showed improved phagocytosis capacity, demonstrating that the observed sialidase effect was a result of the removal of α2,6-sialic acid. The phagocytosis of different pathogenic E. coli isolates was also enhanced by sialidase, which suggests that modifications on MDDC sialic acids may be considered in the development of MDDC-based antibacterial therapies. Physiologically, our findings shed new light on mechanisms that modulate the function of both immature and mature MDDCs, in the context of host-bacteria interaction. Hence, with particular relevance to DC-based therapies, the engineering of α2,6-sialic acid cell surface is a novel possibility to fine tune DC phagocytosis and immunological potency.

Figure 1

Sialidase treatment improves phagocytosis by human monocyte‐derived dendritic cells (MDDCs) and mature MDDCs. Immature MDDCs and lipopolysaccharide (LPS) ‐matured MDDCs (mMDDCs) were treated with sialidase or left untreated, following incubation with fluorescent Escherichia coli, for 1 hr at 4° or 37°. (a) The phagocytic capacity by MDDCs and mMDDCs was evaluated by flow cytometry as the mean fluorescence intensity (MFI) and values obtained at 4° were subtracted. Values represent the means of at least 20 independent assays. Statistical significance (**P < 0·001 or ***P < 0·0001) refers to the difference between untreated and sialidase‐treated MDDCs or mMDDCs. (b) Representative confocal microscopy images showing MDDCs with internalized E. coli (green). Actin filaments of the MDDCs’ cytoskeleton were stained with Phalloidin Alexa Fluor 633 (red). (c) MDDCs were stained with Sambucus nigra lectin (SNA; recognizing α2,6‐sialic acids) and Maackia amurensis lectin (MAA; recognizing α2,3‐sialic acids) lectins following sialidase treatment and analysed by flow cytometry. Values represent the means of the MFI of at least three independent assays. Statistical significance (*P < 0·05) refers to the difference between untreated and sialidase‐treated MDDCs.

Figure 2

Sialidase affects the cytoskeleton organization and the activation of Rho GTPases. Monocyte‐derived dendritic cells (MDDCs) were treated with sialidase or left untreated, and then incubated or not (control) with Escherichia coli for 15 min at 37°. (a) The MDDC cytoskeleton was stained with Phalloidin Alexa Fluor 633 (red) and analysed by confocal microscopy. (b) The Rho GTPase activation level was measured in MDDC lysates, as described in the Materials and methods section. The level of active Rac1 or Cdc42 (in arbitrary units) is based in optical density values obtained at 490 nm after subtracting negative control values. Values represent the means of at least four independent assays. Statistical significance (*P < 0·05) refers to the difference between control MDDCs and MDDCs incubated with E. coli.

Figure 3

Sialidase treatment improves the immunological function of monocyte‐derived dendritic cells (MDDCs). MDDCs were treated with sialidase or left untreated, and then incubated or not (control) with Escherichia coli. (a) The expression of interleukin‐10 (IL‐10), IL‐12, tumour necrosis factor‐α (TNF‐α) and IL‐6 cytokine genes was evaluated by quantitative real‐time PCR in total RNA extracted MDDCs (following sialidase and 1‐hr incubation with E. coli). The mRNA levels of each cytokine are expressed as the permillage (‰) of the expression of the endogenous positive control, β‐actin. Values represent the means of at least six independent assays. Statistical significance (*P < 0·05) refers to the difference between untreated and sialidase‐treated MDDCs following E. coli phagocytosis. (b) Representative images of the nuclear factor‐κB (NF‐κB) transcription factor nuclear translocation. Translocation was assessed by labelling MDDCs (following sialidase and a 15‐min incubation with E. coli) with anti‐NF‐κB p65 (red) and staining the cell nucleus with DAPI (blue). Cells were then fixed and analysed by combining colours through microscopy. At least 600 cells in each condition were analysed. (c) Interferon‐γ (IFN‐γ) gene expression was evaluated by quantitative real‐time PCR in total RNA extracted from a 48 hr co‐culture of MDDCs, (following sialidase and a 1‐hr incubation with E. coli) and autologous T lymphocytes, in a DC : T‐lymphocyte ratio of 1 : 4. The IFN‐γ mRNA levels are expressed as the permillage (‰) of the expression of the endogenous positive control, β‐actin. Values represent the means of at least seven independent assays. Statistical significance (*P < 0·05) refers to the difference between untreated and sialidase‐treated MDDCs following E. coli phagocytosis.

Figure 4

Escherichia coli phagocytosis is influenced by sialic acid moieties. Monocyte‐derived dendritic cells (MDDCs) were treated with sialidase or left untreated, and then incubated or not (control) with E. coli for 1 hr at 4° or 37°. (a) The influence of free sialic acid on phagocytosis was determined by evaluating the phagocytic capacity of MDDCs in the presence or absence of this sugar. Values, representing phagocytosis, calculated as the percentage (%) of the mean fluorescence intensity (MFI; obtained by flow cytometry) normalized with respect to untreated MDDCs incubated without free sialic acid. Values represent the means of at least five independent assays. Statistical significance (**P < 0·001) refers to the difference between sialidase‐treated and untreated MDDCs. (b) The effect of hiding surface α2,6‐linked and α2,3‐linked sialic acids in phagocytosis was determined by incubating untreated MDDCs in the absence (control) or in the presence of Sambucus nigra lectin (SNA) or Maackia amurensis lectin (MAA) blocking lectins. Values represent the means of MFI of at least three independent assays. (c) The influence of ectosialyltransferase activity in phagocytosis was assessed by conducting the assay in the presence or absence of the sialyltransferase substrate, cytidine 5′‐monophospho‐N‐acetylneuraminic acid (CMP‐5‐NeuAc). Values represent the means of MFI of at least five independent assays. Statistical significance (**P < 0·001) refers to the difference between sialidase‐treated and untreated MDDCs. (d) The participation of sialic acids from E. coli surface on phagocytosis was determined by comparing the capacity of MDDCs to internalize E. coli when sialidase‐treated or left untreated. Values represent the means of MFI of at least three independent assays. Statistical significance (**P < 0·001) refers to the difference between sialidase‐treated and untreated MDDCs.

Figure 5

Sialidase treatment improves the capacity of monocyte‐derived dendritic cells (MDDCs) to phagocytose pathogenic Escherichia coli isolates. MDDCs were sialidase‐treated or left untreated and incubated for 1 hr, at 4° or 37°, with pathogenic E. coli isolates (I, II, III, IV). The phagocytic capacity was evaluated by flow cytometry as the MFI and values obtained at 4° were subtracted. Values represent the means of at least 10 independent assays. Statistical significance (**P < 0·001 or ***P < 0·0001) refers to the difference between sialidase‐treated and untreated MDDCs.

Figure 6

Siat1‐null bone marrow‐derived dendritic cells (BMDCs) have improved phagocytic capacity compared with wild‐type (WT). BMDCs were harvested from Siat1‐null and WT mice and incubated in presence of Escherichia coli, for 1 hr, at 4° and 37°. The data represent the means of the MFI values, obtained by flow cytometry; of at least four independent assays (values obtained at 4° were subtracted). Statistical significance (***P < 0·0001) refers to the difference between WT and Siat1‐null BMDCs.