MALDI-TOF MS monitoring of PBMC activation status in sepsis

Abstract

Background: MALDI-TOF mass spectrometry (MS) on whole cells enables the detection of different cell types and cell activation. Here, we wondered whether this approach would be useful to investigate the host response in sepsis.

Methods: Peripheral blood mononuclear cells (PBMCs) from patients with severe sepsis and healthy donors were analyzed with MALDI-TOF MS. PBMCs from healthy donors were also stimulated with lipopolysaccharide, peptidoglycan, CpG oligonucleotides, polyinosinic polycytidylic acid, and with heat-inactivated bacteria. Averaged spectra of PBMCs stimulated in vitro by different agonists were generated from the database using the Biotyper software and matching scores between each spectrum from patients and averaged spectra from the database were calculated.

Results: We show that the MALDI-TOF MS signature of PBMCs from septic patients was specific, compared with healthy controls. As the fingerprints observed in patients may be related to PBMC activation, PBMCs from healthy controls were stimulated with cytokines, soluble Pathogen-Associated Molecular Patterns (PAMPs) and heat-killed bacteria, and we created a database of reference spectra. The MALDI-TOF MS profiles of PBMCs from septic patients were then compared with the database. No differences were found between patients with documented infection (n = 6) and those without bacteriological documentation (n = 6). The spectra of PBMCs from septic patients matched with those of interferon-γ- and interleukin-10-stimulated PBMCs, confirming that sepsis is characterized by both inflammatory and immunoregulatory features. Interestingly, the spectra of PBMCs from septic patients without documented infection matched with the reference spectrum of PBMCs stimulated by CpG oligonucleotides, suggesting a bacterial etiology in these patients.

Conclusions: Despite the limits of this preliminary study, these results indicate that the monitoring of functional status of PBMCs in peripheral blood by whole cell MALDI-TOF MS could provide unique opportunities to assess disease progression or resolution in clinical settings.

Keywords: CpG oligonucleotides; IFN-γ; Interleukin-10; MALDI-TOF; Mass spectrometry; Mononuclear cells; Sepsis.

Fig. 1

MALDI-TOF MS spectra of PBMCs. The PBMCs (1 × 10⁶ cells) from ten healthy donors (a) and ten septic patients (b) were suspended in 10 μL of PBS, and 1 μL was deposited onto the MALDI target. Representative MALDI-TOF MS spectra are shown. MALDI-TOF MS spectra were analyzed using statistical analysis software R (version 2.13)

Fig. 2

Dendrogram representation of PBMCs. The dendrogram constructed by Ouedraogo et al. [17] was implemented with reference spectra of PBMCs from two healthy donors and two septic patients. The Biotyper (Bruker Daltonics) software was used to create an averaged spectrum for each patient, corresponding to at least 10 individual spectra. The averaged spectra were added to the database using the Biotyper software and the dendrogram creation method. Peripheral blood mononuclear cells (PBMCs); polymorphonuclear cells (PMNs); dendritic cells (DCs); monocyte-derived macrophages (MDMs); bone marrow-derived macrophages (BMDMs); red blood cells (RBCs)

Fig. 3

Gel view representation of activated PBMCs. The PBMCs from healthy donors were stimulated with 20 ng/ml IFN-γ, IL-4, IL-10 or 1 μg/mL LPS for 18 h. The spectra were arranged in a pseudo-gel format using a gel view representation. Vertical axis refers to the m/z ratio. Spectra are classified according to the presence/absence of peaks. Unstimulated PBMCs are presented in white

Fig. 4

Hierarchical clustering of activated PBMCs. PBMCs were stimulated with different agonists for 18 h. The results are shown as hierarchical clustering. Vertical axis refers to the m/z ratio. Spectra are classified according to the presence/absence of peaks. PBMCs stimulated with LPS from Escherichia coli are presented in yellow, with Pseudomonas aeruginosa (P.a) in red, Escherichia coli (E.c) in orange, CpG ODN in dark green, PGN in green, Staphylococcus aureus (S.a) in blue, Streptococcus agalactiae (S.ag) in turquoise, unstimulated PBMCs in purple and PBMCs stimulated with poly I:C in dark blue

Fig. 5

Comparison between in vitro and in vivo data. Averaged spectra of PBMCs stimulated in vitro by different agonists were generated from the database using the Biotyper software. The spectra (n = 16) from four patients with E. coli bacteremia, two patients with S. aureus bacteremia and six patients with undocumented infection were then compared with the averaged spectra of the database. Scatter plots of one representative septic patient with E. coli infection (a), S. aureus infection (b) or without microbiological documentation (c) are presented. Matching scores between each spectrum from patients and averaged spectra from the database are represented with circles. Horizontal lines represent the medians of matching scores; a value higher than 1.5 was considered significant and allowed confident identification of the activation status of PBMCs. The nonparametric Mann-Whitney U test was used to compare scores with the averaged spectra of the database