Monocytes, as innate immune cells, are recruited to sites of infection or injury to modulate immune responses and tissue repair. Ca²⁺, which is released from dying cells, could act as an attractant for monocytes eliciting chemokinetic responses. Triggering further the production and release of pro-inflammatory cytokines, Ca²⁺ mimics a danger signal on monocytes in terms of sterile inflammation. This extracellular signal is transduced through calcium-sensing receptor (CaSR) into immunomodulatory responses, more specifically into the assembly of the inflammasome platform, which promotes the maturation of pro-interleukine-1β and pro-interleukine-18 and subsequently their release (Rossol et al., 2012). Given the ubiquitous presence of Ca²⁺ in the human body and additional microenvironments of elevated [Ca²⁺], e.g., during cell signaling, at erosion zones of osteoclasts, but also in non-normal states like inflammation (e.g. gingivitis), skin injury, and cell death, one may ask how a cell could discriminate between physiological and pathological calcium concentrations. The present study unraveled the mechanism of Ca²⁺-induced inflammasome activation in human monocytes. Calcium and phosphate or rather calciprotein particles were uncovered as the main instigators of inflammation in the present in vitro setup.
The results demonstrated that strong Ca²⁺-mediated pro-inflammatory responses of monocytes are only present under increased extracellular phosphate concentrations. Such cell culture conditions are prone to the formation of calciprotein particles mainly consisting of calcium, phosphate, and fetuin-A. Due to the presence of fetuin-A in fetal (bovine) serum, crystallization of calcium and phosphate is prevented, and the size of the formed amorphous particles remains around 100 nm. The simultaneous existence of Ca²⁺ facilitates the activation of CaSR, which in turn boosts constitutive macropinocytosis. This mechanism is at least partially mediated by Gαq protein-dependent pathways. The increased uptake of extracellular fluid and thereby uptake of calciprotein particles lead to enhanced lysosomal cathepsin B activity, but obviously not to lysosomal membrane leakage, as it is known for crystalline structures. Despite lysosomal membrane integrity, a yet unknown mechanism drives inflammasome activation along with Interleukine-1β (IL-1β) release and cell death. A further role of phosphate in CaSR signaling under the present conditions is discussed, as anion binding sites are already described for this receptor.:I LIST OF ABBREVIATIONS 7
II LIST OF FIGURES 11
III LIST OF TABLES 13
1 INTRODUCTION 14
1.1 Ca²⁺ and Pi, an indispensable, but dangerous team 14
Physiological relevance 14
Regulation of Ca²⁺ homeostasis 14
Regulation of [Pi] homeostasis 15
Clinical relevance 16
Calciprotein particle (CPP) 17
1.2 The extracellular calcium ion-sensing receptor - CaSR 19
General overview 19
CaSR-dependent macropinocytosis 21
1.3 Monocytes – mononuclear phagocytes 23
1.4 The inflammasome, activation and physiological function 25
NLRP3 inflammasome 25
Ca²⁺-/CPP-triggered activation of NLRP3 inflammasome 27
1.5 Objective 29
2 MATERIAL AND METHODS 30
2.1 Material 30
2.1.1 Human material 30
2.1.2 Cell lines 30
2.1.3 Media, buffer, chemicals, and consumables 30
2.1.4 Antibodies 31
2.1.5 Assay Kits 31
2.2 Methods 32
2.2.1 Isolation and culture of monocytes from human blood 32
2.2.2 Cell culture and transfection of cell lines 33
2.2.3 [Ca2+] measurement in cell culture medium 33
2.2.4 Dynamic mass redistribution (DMR) measurement 33
2.2.5 CPP preparation and stimulation of monocytes with CPPs 34
2.2.6 Detection of macropinocytosis 35
2.2.7 Detection of cathepsin activity 35
2.2.8 Detection of lysosomal leakage 36
2.2.9 Detection of depolarization of mitochondria 36
2.2.10 Detection of cell death 36
2.2.11 Live-imaging confocal Raman microspectroscopy 37
2.2.12 Transmission electron microscopy - electron dispersive x-ray analysis 37
2.2.13 CPP analysis via dynamic light scattering (DLS) 39
2.2.14 Detection of IL-1β via ELISA 39
2.2.15 Detection and quantification of intracellular cAMP 40
2.2.16 Western Blot 41
2.2.17 Statistics 41
3 RESULTS 42
3.1 Ca²⁺ stimulates NLRP3 inflammasome activation through CaSR signaling only at elevated [Pi] 42
3.1.1 [Pi] > 3 mM is necessary for Ca²⁺-triggered IL-1β release 42
3.1.2 NLRP3 is necessary for Ca²⁺-triggered IL-1β release 43
3.1.3 CaSR partially mediates Ca²⁺-triggered NLRP3 inflammasome activation 44
3.1.4 Pi is not replaceable during Ca²⁺-triggered IL-1β release 45
3.2 Ca²⁺-activated GPCR signaling is affected by Pi 46
3.2.1 Ca²⁺-triggered signaling differs between high and low [Pi] cell culture conditions in monocytes 46
3.2.2 CaSR-mediated DMR responses are affected by Pi 51
3.3 Addition of Ca²⁺ to serum-containing RPMI1640 cell culture medium results in spontaneous formation of nanoparticles 55
3.4 Ca²⁺ stimulation of monocytes enhances macropinocytosis 60
3.4.1 Visualization of CPP uptake 60
3.4.2 CPPs were taken up by an actin- and CaSR-dependent mechanism 64
3.5 Actin remodeling is necessary for IL-1β production after stimulation with [Ca²⁺] in high [Pi] medium 69
3.5.1 Actin remodeling is necessary for Ca²⁺-triggered IL-1β production 69
3.5.2 CPPs and elevated extracellular [Ca²⁺] are responsible for IL-1β release of human monocytes 70
3.5.3 Sr²⁺ prevents Ca²⁺-triggered inflammasome activation 75
3.6 Formation and uptake of CPPs results in increased cathepsin B activity and non-apoptotic cell death 78
3.6.1 Elevated [Ca²⁺] and [Pi] lead to increased cathepsin B activity 78
3.6.2 Ca²⁺-induced cell death strongly depends on elevated [Pi] 81
3.6.3 Cell death (LDH release) partially depends on NLRP3 and CaSR 84
3.7 Summary and graphical overview of results 85
4 DISCUSSION 86
4.1 CaSR is involved in Ca²⁺-mediated NLRP3 inflammasome activation 88
4.2 [Pi] influences Ca²⁺-induced cellular reactions 90
4.2.1 Effects of [Pi] on extracellular calcium ion-sensing receptor signaling 90
4.3 Uptake of CPPs triggers inflammasome activation and is accompanied by cell death 94
4.3.1 Fetuin-A, [Ca²⁺], and [Pi] determine CPP formation and pro-inflammatory responses 94
4.3.2 Effects of CaSR agonists and Gαq-activating GPCR ligands on macropinocytosis and inflammasome activation 97
4.3.3 The inhibitory effects of Sr²⁺ in Ca²⁺-mediated inflammasome activation 98
4.3.4 Uptake of CPPs is accompanied by increased lysosomal activity 100
4.3.5 IL-1β release and cell death are mediated by lysosomal Ca²⁺ efflux 101
4.3.6 Pi fluxes are potentially involved in inflammasome activation 102
4.3.7 Potential mediators of cell death 103
4.4 Clinical relevance of CPPs 105
4.5 Outlook 108
5 SUMMARY 110
6 REFERENCES 114
7 SUPPLEMENTARY 131
7.1 Supplementary figures 131
7.2 Supplementary tables 135
8 DECLARATION OF AUTHORSHIP 146
LIST OF PUBLICATIONS AND TALKS 147
ACKNOWLEDGEMENT 148
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:70644 |
| Date | 27 April 2020 |
| Creators | Jäger, Elisabeth |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English, German |
| Detected Language | English |
| Type | info:eu-repo/semantics/acceptedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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