title: Formyl peptide receptors as druggable targets in viral infections
Creators: name: Baum, Lisa Sandra Valerie
Publisher: name: University of Muenster, Institutional Repository MIAMI
Year: 2025
Subjects: subject: 570 Biology

description: Diese Studie untersucht die Rolle von alveolären Makrophagen (AMs) und ihren Formylpeptid-Rezeptoren (Fprs) bei viralen Lungeninfektionen mit Influenza-A-Virus (IAV). Eine murine ex vivo Makrophagen-ähnliche Zelllinie (AML), die AMs auf Transkriptom- und Proteom-Ebene ähnelt, wurde etabliert und zeigte Reaktionen auf verschiedene Stimuli. Die AML-Zellen exprimierten Fpr1 und Fpr2, welche die extrazellulär-signalregulierten Kinasen 1/2 aktivierten. Obwohl Fprs keine Wirkung bezüglich der Polarisierung der AMs zeigten, beeinflussten sie die IAV-Infektion stammspezifisch. Die Studie untersuchte auch die Fpr-unabhängigen Eigenschaften des Fpr Liganden Annexin A1 (AnxA1), wessen Bindung an Phosphatidylserin kooperativ durch Cholesterin und Ceramid beeinflusst wurde. Die Ergebnisse dieser Arbeit deuten darauf hin, dass Fprs und ihre Liganden AMs bei viralen Lungenkrankheiten modulieren und potenziell neue, wirtsgerichtete therapeutische Ansätze für IAV-Infektionen sein könnten.
description: This study investigates the role of alveolar macrophages (AMs) and their formyl peptide receptors (Fprs) in viral lung infections, focusing on influenza A virus (IAV). A murine ex vivo alveolar macrophage-like (AML) cell line, resembling AMs at the transcriptomic and proteomic levels, was established and shown to respond to various stimuli. AML cells expressed Fpr1 and Fpr2, signaling through extracellular-signal regulated kinase (ERK) 1/2 and not cyclic adenosine monophosphate. While Fprs were not involved in AM polarization, Fpr-induced ERK signaling influenced IAV infection in a strain-specific manner. The study also explored the Fpr-independent lipid-binding properties of the Fpr ligand Annexin A1 (AnxA1), finding that its binding to phosphatidylserine was cooperatively influenced by cholesterol and ceramide. These results suggest that Fprs and their ligands modulate AMs in viral lung diseases, potentially guiding the development of novel host-targeted therapeutic strategies for IAV infections.
description: TABLE OF CONTENTS ..... I
SUMMARY ..... IV
1 Introduction ..... 1
1.1 The vertebrate immune system ..... 1
1.2 Macrophages as key players in the inflammatory response ..... 2
1.3 Alveolar macrophages ..... 3
1.4 Murine macrophage polarization ..... 5
1.5 AMs and pulmonary influenza A virus infections ..... 9
1.6 Formyl peptide receptors and their role in macrophages ..... 12
1.6.1 FPR signal transduction ..... 13
1.6.1.1 The cAMP signalling pathway ..... 14
1.6.1.2 The MAPK/ERK signalling cascade ..... 15
1.7 Mammalian toll-like receptors ..... 18
1.8 Annexin A1, a member of the annexin family ..... 21
1.8.1 AnxA1 in immune regulation ..... 21
1.8.2 AnxA1 and biological membranes ..... 23
2 Aims of this study ..... 27
3 Materials ..... 29
3.1 Chemicals and Reagents ..... 29
3.2 Lab ware ..... 32
3.3 Kits ..... 33
3.4 Media and buffers ..... 33
3.4.1 Self-made media and buffers ..... 34
3.5 Primers ..... 34
3.5.1 Quantitect primer assays (Qiagen) ..... 34
3.5.2 Microsynth primer assays ..... 35
3.6 Vectors ..... 35
3.7 Antibodies and cell stains for FACS and Céligo ..... 36
3.8 Laboratory devices ..... 37
3.9 Bacteria, cell lines, and viruses ..... 38
3.10 Computer Software ..... 38
4 Methods ..... 39
4.1 Cell culture and stimulation ..... 39
4.1.1 Culture of MDCK cells ..... 39
4.1.2 Culture of MPI cells ..... 39
4.1.3 Reception of progenitor cells from murine bone marrow ..... 40
4.1.4 Macrophage generation from murine bone marrow progenitor cells ..... 40
4.1.4.1 Generation of BMDM from murine bone marrow monocytes ..... 40
4.1.4.2 Generation and culture of AML cells ..... 41
4.1.5 Macrophage polarization and stimulation ..... 42
4.2 Flow cytometry ..... 42
4.3 HTRF-FRET-based cAMP quantification ..... 43
4.4 HTRF-FRET-based quantification of ERK phosphorylation ..... 44
4.5 Transcriptomic analysis ..... 44
4.5.1 RNA extraction ..... 44
4.5.2 cDNA Synthesis ..... 45
4.5.3 Reverse transcription quantitative realtime PCR ..... 45
4.6 Virology ..... 46
4.6.1 IAV infections ..... 46
4.6.2 Virus staining, detection and infection analysis ..... 46
4.7 QCM-D Measurements ..... 47
4.7.1 Lipid preparation ..... 47
4.7.2 Preparation of QCM-D measurements ..... 48
4.7.3 QCM-D-measurements and analysis ..... 49
4.8 Data analysis ..... 50
5 Results ..... 51
5.1 Part I: Fprs, AnxA1 and viral infections in murine AMs ..... 51
5.1.1 AML cells as a model system for murine AMs ..... 51
5.1.1.1 Establishment and verification of an ex vivo AM cell line ..... 52
5.1.1.2 AM marker exposition by AML cells and differentiation from BMDMs ..... 53
5.1.1.3 AML cells can be polarized into M1, M2a and M2c macrophages ..... 55
5.1.1.3.1 Transcriptomic verification of AML cell polarization ..... 56
5.1.1.3.2 Proteomic verification of AML cell polarization ..... 60
5.1.2 The role of Fprs in AML cells ..... 62
5.1.2.1 Transcriptomic analysis of Fpr-related genes in AML cell polarization ..... 62
5.1.2.2 AML cell differentiation and polarization are rather Fpr-independent ..... 65
5.1.2.3 Gαs signalling affects AML cell polarization ..... 72
5.1.2.4 AML cell polarization does not affect intracellular cAMP levels ..... 73
5.1.2.5 Adenylate cyclase expression in untreated and polarized AML cells ..... 74
5.1.2.6 Fpr agonist-induced ERK1/2 phosphorylation in AML cells ..... 77
5.1.2.7 TLRs: Another group of PRRs and their role in AML cell polarization ..... 80
5.1.2.7.1 TLR mRNA expression changes upon polarization ..... 80
5.1.2.7.2 Stimulation of AML cells with TLR ligands ..... 82
5.1.3 The interplay of annexinA1, Fpr stimulation and viral infections in AMs ..... 84
5.1.3.1 AML cells are susceptible to infection with IAV pdm09 and SC35M ..... 84
5.1.3.2 Polarization of AML cells affects the infection success of IAV ..... 88
5.1.3.3 AnxA1 virus strain-specifically affects IAV infection of AML cells ..... 89
5.1.3.4 Fpr signalling affects IAV infection success in AML cells ..... 90
5.2 Part II: Membrane binding of AnxA1 in the presence of Cer and Chol ..... 92
5.2.1 Establishment of bilayers for QCM-D measurements ..... 92
5.2.2 The dynamic binding of AnxA1 depends on POPS and Ca2+ ..... 93
5.2.3 AnxA1 binding to artificial cell membranes with low Cer content ..... 95
5.2.4 The combination of Cer and Chol cooperatively influences AnxA1 binding ..... 97
5.2.5 The cooperative effect of Chol is stronger for AnxA2 than for AnxA1 ..... 98
6 Discussion ..... 101
6.1 AML cells as an in vitro model of murine AMs ..... 101
6.2 Fpr-mediated signalling in murine AMs ..... 104
6.3 TLR-mediated signalling in murine AMs ..... 113
6.4 The role of AM stimulation and Fpr signalling in IAV infections ..... 117
6.5 Fpr-independent binding of AnxA1 in the presence of Cer and Chol ..... 125
6.6 Conclusions and outlook ..... 129
7 Supplements ..... 132
7.1 The role of Fprs in AML cells ..... 132
7.1.1 Cell sizes and FACS profile of murine macrophage cell lines ..... 132
7.1.2 FACS histograms of the F4/80 exposition in AML- and BMDM cells ..... 133
7.1.3 Differentiation of AML cells after 9 days of culture [%] ..... 133
7.1.4 Phenotypes of polarized AML cells as observed by light microscopy ..... 134
7.1.5 Ct values of all genes in WT AML cells ..... 134
7.1.6 Isotype controls of antibodies for the detection of AM polarization ..... 135
7.1.7 Fpr1KO and Fpr2KO AML cell polarization, FACS analysis ..... 135
7.1.8 Marker gene Ct values of UT WT, Fpr1KO and Fpr2KO AML cells ..... 136
7.1.9 DMSO affects AML cell polarization ..... 137
7.1.10 Ct values of ACs in unstimulated WT AML cells ..... 137
7.1.11 ERK phosphorylation in AML cells, non-transformed and cell line-specific ..... 138
7.1.12 HTRF ratio changes in AML cells upon PMA stimulation ..... 139
7.1.13 Ct values of TLRs in untreated WT AML ..... 140
7.1.14 AML cell infection with IAV ..... 140
7.2 QCM-D ..... 141
7.2.1 Overview on previous work ..... 141
7.2.2 Annexin synthesis ..... 141
7.2.2.1 Annexin production in E. coli DH5α ..... 142
7.2.2.2 Cellular disruption and annexin purification ..... 142
7.2.2.3 Annexin detection via SDS-PAGE and western blot ..... 143
7.2.3 Lipid compositions of simple and complex bilayers ..... 145
7.2.4 Dependency of ΔΔD5 and ΔΔF5 for equilibrium frequency shifts of the binding
isotherms ..... 146
BIBLIOGRAPHY ..... V
LIST OF ABBREVIATIONS ..... XXXIV
ACKNOWLEDGEMENTS ..... XXXVIII
EIDESSTATTLICHE ERKLÄRUNGEN ..... XXXIX
CURRICULUM VITAE ..... XL