Phosphoproteome profiling of the macrophage response to different toll-like receptor ligands identifies differences in global phosphorylation dynamics

Abstract

Toll-like receptors (TLRs) are among the first sensors that detect infection and drive immune response. Macrophages encountering a pathogen are usually stimulated not by one TLR, but by a combination of TLRs engaged by distinct microbe ligands. To understand the integrated signaling under complex conditions, we investigated the differences in the phosphoprotein signaling cascades triggered by TLR2, TLR4, and TLR7 ligands using a single responding cell population. We performed a global, quantitative, early poststimulation kinetic analysis of the mouse macrophage phosphoproteome using stable isotope labeling with amino acids coupled to phosphopeptide enrichment and high-resolution mass spectrometry. For each TLR ligand, we found marked elevation of phosphorylation of cytoskeleton components, GTPases of the Rho family, and phospholipase C signaling pathway proteins. Phosphorylation of proteins involved in phagocytosis was only seen in response to TLR2 and TLR4 but not to TLR7 activation. Changes in the phosphorylation of proteins involved in endocytosis were delayed in response to TLR2 as compared to TLR4 ligands. These findings reveal that the phosphoproteomic response to stimulation of distinct TLRs varies both in the major modification targets and the phosphorylation dynamics. These results advance the understanding of how macrophages sense and respond to a diverse set of TLR stimuli.

Keywords: SILAC; TLR2; TLR4; TLR7; innate immunity; macrophage; phosphoproteomics; toll-like receptors.

Figure 1

Experimental system and design. (A) Strategy for global and quantitative analyses of LPS-, P3C-, and R848-induced phosphorylation. C57 derived macrophages were SILAC labeled with normal or stable isotope-substituted arginine and lysine amino acids resulting in three states distinguishable by their mass. Each cell population was left untreated or stimulated for 3, 5, 10, or 30 min. The 10 min stimulation time point was included in both pools to serve as a common reference point. Cell lysates to be directly compared were pooled, enzymatically digested, and fractionated by SCX. The phosphopeptides were enriched by TiO₂ and analyzed by online LC–MS(/MS). The mass shift introduced by the SILAC amino acids resulted in triplet peaks (i.e., the same peptide from three different time points) with the relative intensities equal to the relative abundance of the peptide. This SILAC approach allows for high-accuracy quantification of phosphopeptides with in most cases localization of the phosphate group with single amino acid accuracy. Two biological replicates were used to perform independent experiments for each ligand stimulation. (B) Labeling efficiency example of a peptide containing a lysine and arginine residue. The arrows indicate the position of partially labeled peptides.

Figure 2

Global comparison of the phosphorylation events that resulted from different ligand stimulation. (A) Overlap of phosphorylation sites from the two independent experiments for each ligand stimulation. Depicted are the phosphorylation sites that were quantified relative to unstimulated macrophages. The downstream bioinformatics analyses used only the reproducibly identified phosphorylation sites. (B) Distribution of phosphorylated amino acids. The total number of quantified phospho-serine (white), phospho-threonine (gray), and phospho-tyrosine (black) sites for each ligand stimulation is indicated. (C) Distribution of the phosphorylated proteins within cellular compartments. The number of proteins within each cellular compartment (black) was compared to the number of phosphoproteins obtained from each ligand stimulation (red: LPS, blue: P3C, purple: R848). Significantly over-represented GO terms are marked with asterisks, and under-represented GO terms are marked with a hash. (D) The absence of TLR4 expression affects cellular migration. C57 derived macrophages or TLR4 knock out stable transfectants in a C57 derived macrophages background were assayed in the presence or absence of 100 ng/mL LPS. Four different cell combinations were used; C57 cells were seeded in the microchamber in the presence of C57 (C57/C57), or C57 TLR4^–/– cells (C57/TLR4^–/–) in the well, or C57 TLR4^–/– cells were seeded in the microchamber in the presence of C57 (TLR4^–/–/C57) or C57 TLR4^–/– (TLR4^–/–/TLR4^–/–) cells in the well. Cell migration was allowed to proceed for 12 h at 37 °C, and cells that transmigrated across the membrane were counted; *p < 0.005 when compared to the wild type C57 derived macrophages. (E) Delay in phosphorylation of phosphoproteins involved in the MyD88 dependent pathway (ERK1, AP-1), which responded earlier to the LPS stimuli compared to the MyD88 independent pathway (TAK1, IRF3), which is activated after TLR4 internalization. NFKBIB is an inhibitor of the MyD88 dependent pathway, and its phosphorylation event coincided with the start of the MyD88 independent pathway. The dotted lines represent significance thresholds (log fold change = ± 0.114). Experiments were performed in duplicate.

Figure 3

Temporal phosphorylation dynamics profiles. (A–E) Five distinct time-course clusters identified by fuzzy c-means clustering of the time series data for all ligands combined. The five selected phosphopeptides and their respective ligand in F–J are typical of the five clusters found in panels A–E. Some phosphopeptides were found in different clusters after stimulation (an example is depicted in panel K), whereas some phosphopeptides clustered together irrespective of the ligand used for their stimulation (an example is depicted in panel L).

Figure 4

Phosphosites common for LPS and P3C (A), LPS and R848 (B), and P3C and R848 (C). Among the phosphosites shared between pairs of ligand stimulations, the number of phosphosites that are nonregulated, regulated, and differentially regulated is indicated.

Figure 5

Induction of phagocytosis in response to LPS but not to R848 (A, B). Increase in phosphorylation of FcγR1 following the LPS stimulation but not R848 stimulation (A) and of Corola (B) post-LPS and P3C but not post-R848 stimulation (B). Both proteins are involved in phagocytosis. (C, D): Delay in endocytosis after P3C stimulation vs. LPS stimulation. Average of the phosphorylation levels for each time point, for phosphosites in proteins classified as involved in endocytosis by the IPA analysis (C). Representative phosphorylation trend of a phosphosite on NECAP2, a protein involved in endocytosis post LPS, P3C, and R484 stimulation (D). (E) Phagocytosis assays of IgG-coated sheep red blood cells (SRBCs). C57 derived macrophages were cultured in 96-well plates with 2000 IgG treated or nontreated SRBCs for 30 min with either no treatment (black bar) or in the presence of 100 ng/mL LPS (horizontal stripes), 1 μM P3C (vertical stripes) or 1 μM R848 (white bar). Phagocytosis was assessed by measuring the absorbance at 620 nm as described in materials and methods. **p < 0.0001, *p < 0.001 when compared to no treatment.