Analysis and comparison of the glycoproteomic phenotype of TLR4- and TLR2-induced tolerance in human monocytes / von Andrea Behnert

Endotoxin tolerance of human monocytes contributes to sepsis-induced immunosuppression, a leading cause of sepsis-related deaths worldwide. Although several studies already demonstrated distinct alterations of cytokine expression, phagocytotic capability and expression changes of particular receptor- and glycoproteins to be linked with the tolerant state in human monocytes, no study investigated changes of the whole glycoproteome on a global level. Due to the fact that most membrane-bound receptors are glycosylated, the characterization of the tolerant monocyte cell state by glycoproteomics using tandem mass spectrometry (LC-MS/MS) might reveal new and useful markers to distinguish tolerant from naϊve cells and can provide possible new targets for improving immune-modulatory therapies. In this doctoral thesis, (a) PAMPs binding to cell surface-expressed pattern recognition receptors (PRRs) (TLR4 agonist LPS, TLR2/1 agonist Pam3CSK and TLR2/6 agonist MALP2), (b) PAMPs binding to intracellular PRR (NOD- like receptor (NLR) ligands 1 and 2; iE-DAP and MDP) and (c) the alarmin S100A12, signaling via the receptor for advanced glycation endproducts (RAGE) and TLR4, were examined for their capability to induce the monocyte tolerant state. Qualitative and quantitative analysis of glycoprotein expression changes in purified monocytes of three different peripheral blood donors were assessed with and without LPS-, Pam3CSK- and MALP2-stimulation and analyzed in the naϊve and tolerant state. Tolerance was measured by restimulation with LPS of monocytes that were prestimulated with adjusted concentrations of either LPS, Pam3CSK or MALP2. Prestimulation with all three PRR agonists led to highly decreased expression of the pro-inflammatory cytokines TNF-α and IL-6, which is consistent with the cells entering a tolerant state. NLR ligands iE-DAP and MDP induced only weak pro-inflammatory responses in human monocytes and none of the NLR ligands demonstrated a reduction of TNF-α expression in subsequent LPS challenges. Commercially available S100A12, that was efficient in inducing pro-inflammatory activation of monocytic THP-1 cells, was found to be significantly contaminated with LPS, as revealed by Limulus Amebocyte-Lysate (LAL) test. Further experiments with endotoxin-free, granulocyte-derived, purified S100A12 induced neither a pro-inflammatory activation of human monocytes nor tolerance. Thus, only the glycoproteomes of LPS-, Pam3CSK- and MALP2-stimulated monocytes, that induced the tolerant state, were analyzed in a mass spectrometry approach. Comparable numbers of glycoproteins (1003, 966 and 1033) were identified in purified human monocytes from each of the three donors, respectively. Altogether, 1176 annotated proteins were identified, originating from various cellular organells. 898 of the 1176 identified glycoproteins were predicted to contain at least one transmembrane domain, demonstrating that a high number of membrane spanning glycoproteins has been found. The majority of the identified glycoproteins were annotated in Gene Ontology (GO) as “plasma membrane” associated including 202 CD antigens and 54 GPCRs. Stimulation of the purified human monocytes with LPS, Pam3CSK or MALP2 induced significantly differential expression levels of 135 glycoproteins. From 75 glycoproteins annotated to be involved in glycan processing and maturation, only 4 demonstrated significantly expression changes, indicating no major changes in the glycosylation of the proteins which might have affected enrichment and quantification during LC-MS/MS analysis. The largest subset of differentially expressed glycoproteins was again annotated as plasma membrane-resident glycoproteins comprising 35 significantly significantly regulated CD antigens. regulated CD antigens.

Saved in:
Person: Behnert, Andrea [Author]
Corporate Author: Friedrich-Schiller-Universität Jena [Degree granting institution]
Format: Book
Language(s):English
Language note:Zusammenfassungen in deutscher und englischer Sprache
Publication:Jena, 2018
Printing place:Jena
Dissertation Note:Dissertation, Friedrich-Schiller-Universität Jena, 2018
Subjects:Sepsis > Monozyt > Immunsuppression > Glykoproteine
Type of content:Hochschulschrift
Related resources:Erscheint auch als Online-Ausgabe: Analysis and comparison of the glycoproteomic phenotype of TLR4- and TLR2-induced tolerance in human monocytes
Physical description:116 Blätter : Illustrationen, Diagramme ; 29,5 cm
Basic Classification: 44.75 Infektionskrankheiten, parasitäre Krankheiten
44.45 Immunologie