Spatiotemporal Changes of the Phagosomal Proteome in Dendritic Cells in Response to LPS Stimulation

Abstract

Dendritic cells (DCs) are professional phagocytes that use innate sensing and phagocytosis to internalize and degrade self as well as foreign material, such as pathogenic bacteria, within phagosomes. These intracellular compartments are equipped to generate antigenic peptides that serve as source for antigen presentation to T cells initiating adaptive immune responses. The phagosomal proteome of DCs is only partially studied and is highly dynamic as it changes during phagosome maturation, when phagosomes sequentially interact with endosomes and lysosomes. In addition, the activation status of the phagocyte can modulate the phagosomal composition and is able to shape phagosomal functions.In this study, we determined spatiotemporal changes of the proteome of DC phagosomes during their maturation and compared resting and lipopolysaccharide (LPS)-stimulated bone marrow-derived DCs by label-free, quantitative mass spectrometry. Ovalbumin-coupled latex beads were used as phagocytosis model system and revealed that LPS-treated DCs show decreased recruitment of proteins involved in phagosome maturation, such as subunits of the vacuolar proton ATPase, cathepsin B, D, S, and RAB7. In contrast, those phagosomes were characterized by an increased recruitment of proteins involved in antigen cross-presentation, e.g. different subunits of MHC I molecules, the proteasome and tapasin, confirming the observed increase in cross-presentation efficacy in those cells. Further, several proteins were identified that were not previously associated with phagosomal functions. Hierarchical clustering of phagosomal proteins demonstrated that their acquisition to DC phagosomes is not only dependent on the duration of phagosome maturation but also on the activation state of DCs. Thus, our study provides a comprehensive overview of how DCs alter their phagosome composition in response to LPS, which has profound impact on the initiation of efficient immune responses.

Keywords: Cell biology*; Cellular organelles*; Endocytosis*; Immunology*; Inflammatory response; Label-free quantification; Protein Identification*; dendritic cell; phagocytosis; phagosome maturation.

Graphical abstract

Fig. 1.

Label-free quantitative proteomics of DC phagosomes. BMDCs were treated with 100 ng/ml LPS for 16 h, and resting and LPS-treated BMDCs were incubated with OVA-coupled beads for 30 min. Noninternalized beads were removed by several washes, and BMDCs were incubated for 15, 60, 120 min to allow phagosome maturation to occur. After the different chase periods, cells were lysed mechanically and LBPs were isolated on a discontinuous sucrose gradient by ultracentrifugation. Phagosomal proteins were digested with trypsin and analyzed by LC-MS/MS on an Orbitrap Fusion spectrometer. Proteins were quantified using MaxQuant and were considered significantly altered in abundance between samples with a p value < 0.05 and a fold change > 1.5. In the volcano plots, the number of significantly enriched proteins in LBPs of resting BMDCs (blue) and in LBPs of LPS-treated BMDCs (orange) is shown. Enrichment of biological processes was determined by gene ontology analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID). LPS: lipopolysaccharide; OVA: ovalbumin; LBP: latex bead-containing phagosome.

Fig. 2.

Kinetics of differential acquisition of phagosomal proteins on LBPs of resting and LPS-treated BMDCs. Differential abundance of phagosomal proteins, grouped according to their function, is shown for three time points (15, 60, and 120 min) during phagosome maturation. Differential acquisition of phagosomal proteins on LBPs isolated from resting and LPS-treated BMDCs is represented by their log2 ratio [LPS/resting]. Enrichment in LBPs of resting BMDCs is shown in blue, whereas increased abundance in LBPs of LPS-treated BMDCs is shown in orange.

Fig. 3.

Clustering of phagosomal proteins according to the kinetics of their differential acquisition. Phagosomal proteins were clustered based on their differential abundance (log2 ratio [LPS/resting]) at three time points (15, 60, and 120 min) during phagosome maturation. Proteins were grouped in 8 clusters by hierarchical clustering using Perseus, based on k-means of cosine similarity.

Fig. 4.

Confirmation of the presence of phagosomal proteins by Western blotting. LBPs were isolated after a 30 min pulse with OVA-coupled beads, followed by a 15 min, 60 min or 120 min chase period. LBPs were purified by ultracentrifugation on a sucrose gradient, and LBP protein lysates were analyzed together with the total cell lysate (TCL) by SDS-PAGE and Western blotting. A, Analysis of phagosome maturation markers together with a validation of LBP purity demonstrated by the absence of a mitochondrial marker (CYT C), Golgi proteins (GM130 and TGN38) and an ER marker protein (YKT6). B, The presence of novel phagosomal proteins (IRG1, NF-κB2, ZBP1, RAB6a, RAB21 and HOOK3) was also demonstrated by Western blotting. TFR: transferrin receptor; EEA1: early endosomal antigen; RAB: Ras-related protein Rab; CANX: calnexin; LAMP-1: lysosome-associated membrane glycoprotein 1; CATH D: cathepsin D; CYT C: cytochrome C; GM130: golgi matrix protein 130; TGN38: trans-golgi network protein 38; YKT6: synaptobrevin homolog YKT6; IRG1: immune-responsive gene 1; NF-κB2: nuclear factor NF-κB p100 subunit; ZBP1: Z-DNA-binding protein 1; HOOK3: protein Hook homolog 3.