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

Les inflammasomes sont des complexes protéiques impliqués dans l’immunité innée, qui sont activés par de multiples signaux de danger. Des mutations héréditaires des protéines de l’inflammasome peuvent être responsables de son activation excessive et in fine de la survenue de pathologies auto-inflammatoires chez l’être humain. À l’heure actuelle, aucun modèle cellulaire ne permet d’étudier spécifiquement la dynamique d’activation des inflammasomes et de préciser les conséquences des mutations activatrices sur celles-ci. J’ai donc généré un modèle humain macrophagique exprimant une protéine recombinante FLAG3x-ASC endogène, commune aux différents inflammasomes dans la lignée cellulaire humaine monocytaire/macrophagique THP-1. Cette lignée a été générée par édition génique par la technologie CRISPR-Cas9 en utilisant un substrat de recombinaison permettant d’insérer la séquence codant pour le FLAG3X in frame du locus PYCARD codant pour ASC. J’ai pu générer 6 clones FLAG3x-ASC dans la lignée THP-1. Les clones générés ont été validés en confirmant l’expression et la fonctionnalité de la protéine recombinante FLAG3x-ASC et en vérifiant l’absence de mutations indésirables hors-cible générée par la nucléase Cas9. Une fois ce modèle généré, j’ai pu également reproduire un variant génétique du gène NLRC4, protéine sensor de l’inflammasome du même nom, retrouvé chez un patient présentant une maladie auto-inflammatoire. La validation des clones mutant pour NLRC4 est en cours. Ce projet permettra la caractérisation de la dynamique d’activation de l’inflammasome dans un modèle physiologique et pathologique. Ceci permettra une avancée importante dans la compréhension de l’inflammasome et son agrégation ainsi que la régulation de ce complexe face aux signaux de danger. / Inflammasomes are multiproteic complexes that are involved in innate immunity and are activated by multiple signals of dangers. Hereditary mutations in inflammasome components lead to its excessive activation that is responsible for human auto-inflammatory disease. While these mutations are supposed to alter the dynamic of inflammasome activation, there is no current human model allowing the dynamic study of this complex. I generated a human cellular model expressing an endogenous FLAG3x ASC protein, an adaptator protein common to several inflammasomes, in the human monocytic/macrophagic THP-1 cell line. This model was created through CRISPR-Cas9 genome engineering using a recombination template allowing the in frame integration of the sequence encoding the FLAG3X peptide at the PYCARD locus encoding ASC. I generated and validated the expression and the functionality of 6 FLAG3x-ASC THP-1 cell lines. Furthermore, these cell lines are devoided of CRISPR-Cas9 off-target effect. In this model, I further reproduced a genetic variant of the inflammasome component NLRC4 observed in a patient presenting with autoinflammatory manifestation. The functional validation of the FLAG3x-ASC THP-1 harboring the NLRC4 variant is on-going. This project will allow to study the dynamic of the activation of the inflammasome in healthy and pathological conditions. Those results will help refine our comprehension of inflammasome complexation and regulation in response to danger signals.

Despite the availability of effective therapy of hypercholesterolemia and hypertension, cardiovascular mortality continues to be very high in the Western world. Inflammatory changes occurring in the arterial wall as well as in the adipose tissue play a major role in the development of atherosclerosis. Macrophages are involved in the process of atherogenesis as early as atherosclerosis begins to develop, when, still as monocytes, they migrate and adhere to the arterial wall as a result of endothelial activation and stimulation by pro-inflammatory substances. Adipose tissue has long been recognized as an important endocrine organ, with part of adipose tissue made up by a large amount of macrophages capable of producing a large number of pro-inflammatory cytokines, which contribute to the development of low-grade chronic inflammation important in the development of atherosclerosis. In samples of subcutaneous, visceral and perivascular adipose tissue (SAT, VAT, and PVAT, respectively) obtained from healthy subjects (living kidney donors, LKD), we analyzed macrophages and their polarization, gene expression of pro-inflammatory cytokines and the effect of substances released by VAT on the level of monocyte adhesion to the endothelium. In some analyses, we included samples of SAT, VAT and PVAT obtained...

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Chlamydia spp. infect epithelial cells of the respiratory, intestinal, and reproductive tracts. Chlamydial infections in women may lead to pelvic inflammatory disease, ectopic pregnancy, chronic pelvic pain, and infertility. In addition to infecting infection the female reproductive tract (FRT), Chlamydia also infects the gastrointestinal tract (GIT) of animals and humans. In mice Chlamydia muridarum disseminates from the FRT to the GIT via internal routes and in a stepwise manner. Initially Chlamydia spreads from the FRT to infect the FRT-draining iliac lymph nodes (ILNs), then the spleen, and then the GIT. The first step of this dissemination (FRT to ILN) is mediated by tissue CD11c+ DCs. Chlamydia transport from ILN to the spleen is dependent on cell transport and is mediated by sphingosine 1-phosphate (S1P) signaling. The third step of Chlamydia transport from the spleen to the GIT is significantly hindered in splenectomized mice. However, which cells mediate this transportation of the second and the third step remain unknown. Using mouse-specific C. muridarum as a model pathogen we show that following depletion of CD8+ T cells or monocytes, Chlamydia dissemination to the spleen and the GIT is significantly hindered. Furthermore, this study reveals that Chlamydia may infect various cell types which then mediate its dissemination internally. It remains to be determined what role systemic dissemination may have in Chlamydia pathogenesis.

OBJECTIVE. To determine if the levels of circulating myeloid-derived suppressor cells increase with progression of prostate cancer (PCa); to determine if such cells could contribute to the relative inefficiency of PCa immunotherapy. MATERIALS AND METHODS. We analyzed peripheral blood mononuclear cells isolated from untreated PCa patients (uPCa; N=18; mean age±SD: 72.1± 6.9 years), tPCa (N = 22; 72.8 ± 9.8 years) and age matched controls (AMC; N = 12; 68.8 ± 7.5 years). We quantified surface marker phenotype, differentiation potential, effects on T cell proliferation and intracellular cytokines. RESULTS. We observed an unexpectedly high percentage of a type of myeloid-derived suppressor cells, CD14+HLA-DR low/- monocytes, in tPCa (30.7±15.0% of CD14+ cells) relative to AMC (4.1+6.5%, P<0.0001) and uPCa (10.6 ± 14.3%, P = 0.0001). The levels of CD14+ HLA-DR low/- cells were significantly correlated with circulating PSA levels and treatment with LHRH-agonist leuprolide in combination with either an antiandrogen or dexamethasone. Monocytes from tPCa inhibited autologous T cell proliferation statistically significantly more effectively than AMC monocytes and were defective in their ability to differentiate into phenotypically mature dendritic cells. Isolated CD14+HLA-DRlow/- cells expressed higher levels of intracellular interleukin-10 and suppressed T cell proliferation more effectively than isolated CD14+HLA-DR+ cells. CONCLUSIONS. This is the first report of CD14+ cells exhibiting reduced expression of HLADRmolecules in PCa patients. These cells suppress immune cell function in vitro and, plausibly, in vivo, a finding that must be factored into the design of immunotherapy protocols for PCa patients.

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With the introduction of antiretroviral therapy (ART), human immunodeficiency virus (HIV) has progressed to a chronic inflammatory disease with accelerated, subclinical end-organ damage, specifically cardiovascular disease (CVD). People with HIV (PWH) have higher incidence, risk, and mortality from CVD, such as atherosclerosis, diastolic dysfunction, and heart failure. Several recent clinical reports have shown that PWH have a predisposition to developing heart failure with preserved ejection fraction (HFpEF), presenting with pathological concentric hypertrophy, diffuse fibrosis, and diastolic dysfunction. As such, an investigation into immunological and molecular mechanisms promoting pathological changes in the heart is necessary. Recent clinical reports show that people living with HFpEF have elevated plasma osteopontin (Opn), and plasma Opn is a powerful predictor of HFpEF severity, HFpEF-related hospitalizations, and mortality. Second, several animal models of HFpEF phenotypes have suggested Opn is involved in driving or perpetuating diastolic dysfunction and cardiac fibrosis. Therefore, we investigated changes in Opn in a cohort of PWH and two translationally relevant models of HIV infection (non-human primates and humanized mice) to identify the pathological role of Opn in cardiac fibrosis and detail the adjunctive potential of Opn for PWH presenting with HFpEF. In Chapter 2, twenty asymptomatic, antiretroviral-treated women with HIV (WHIV) and fourteen women without HIV (HIV-women) matched on age and body mass index underwent cardiac magnetic resonance imaging (MRI) and immune phenotyping. First, we compared a number of immunological parameters to the extensive cardiac MRI parameters in WHIV. Secondly, we analyzed relationships between plasma Opn with cardiac structure and function and markers of immune activation among WHIV, HIV- women, and the whole cohort. Multivariable modeling among the whole group was performed using myocardial fibrosis and myocardial steatosis, respectively, as the dependent variable and HIV status, atherosclerotic cardiovascular disease (ASCVD) risk score, and plasma Opn as independent variables. Among WHIV, multi-variable modeling was performed using plasma Opn as the dependent variable and CD4+ T cell count, HIV viral load, and the respective immune parameter, relating to plasma OPN in bivariate analyses, as an independent variable. In Chapter 3, we investigated bulk transcriptomic changes in the left ventricle of the heart in a model of HIV infection. We utilize the highly translatable simian immunodeficiency virus (SIV)-infected rhesus macaque model to identify changes in the myocardium with and without ART. Animals were selected by viral load, with SIV-infected animals having a high titer of plasma viral load and the SIV-infected animals with ART having a reduction in viral load by several logs. We performed total RNA-Seq on left ventricle tissue from uninfected animals, SIV-infected animals, and SIV-infected animals receiving a clinically relevant ART regimen. SIV infection led to high plasma viral load, but little to no SIV RNA was detectable in the left ventricle, shown by minimal of SIV RNA+ cells in the heart and no SIV sequences identified from RNA-Seq. SIV infection produced a highly inflammatory reaction in the heart, predominated by interferon and pathogen response. Additionally, interferon gamma (IFNg) and lipopolysaccharide (LPS) were both identified as potential upstream drivers of transcriptomic changes in the heart from SIV infection. Reduction of viral load by ART reduced the interferon and cytokine response in the heart; however, SIV-infected animals receiving ART exhibited decreased expression of integral genes directly involved in fatty acid (FA) metabolism, carnitine shuttling, and beta-oxidation. In Chapter 4, we use both in vitro and in vivo modeling to identify molecular mechanisms involved in the development of cardiac fibrosis. We utilized mouse embryonic fibroblasts (MEFs) modeled cardiac fibroblasts and were stimulated with IFNg, LPS, and TGF-b for phenotypic changes in contraction and cytokine production. The interplay of Opn and cardiac fibrosis was investigated in SIV-infected macaques with/without ART and HIVinfected humanized mice with/without an Opn-inhibiting RNA aptamer. LPS-stimulated MEFs retained myofibroblast-like contractility from TGF-b stimulation, secreted inflammatory cytokines/chemokines, and produced Opn (Spp1 transcripts). SIV-infected animals had elevated plasma Opn at necropsy, accumulation of full-length Opn in the ventricle, and ventricular interstitial fibrosis. Multivariate regression identified growth differentiation factor (GDF)-15, inflammatory CD14+CD16+ monocytes, and CD163 expression on CD14+ CD16+ monocytes as independent predictors of plasma Opn during SIV infection. HIV-infected humanized mice showed increased interstitial fibrosis compared to uninfected/untreated animals, and systemic inhibition of Opn by RNA aptamer reduced left ventricle fibrosis in HIV-infected humanized mice. These studies combined a clinical cohort of WHIV, SIV-infected rhesus macaques, and HIV-infected humanized mice to determine the role of Opn in the pathophysiology of cardiac deficits from HIV infection. Our primary goals were to begin to unravel the role of Opn in the development of HFpEF phenotypes seen in PWH, detail Opn as a converging biomarker of cardiac stress and remodeling and immune dysfunction in HIV infection, and investigate the therapeutic potential of Opn to reduce cardiac fibrosis from HIV infection. While Opn has a broad spectrum of physiological and pathological functions, we aimed to frame Opn as an important protein of interest in future studies into HFpEF in PWH. / Biomedical Sciences

Cardiovascular disease leads in morbidity and mortality in Western societies with no known cure. NADPH oxidase (Nox) contributes to atherosclerosis through the indirect activation of macrophages leading to the internalization of oxidized low density lipoproteins (oxLDL). Chronic inflammation in activated macrophages contributes to atherosclerosis. Because macrophages are positioned at the cross-roads of lipid metabolism in vessel walls, they are important in the cellular pathology of atherosclerosis. Components of the endocannabinoid (eCB) system are vital to atherosclerotic development, since the eCB system has been found to play an important role in the amelioration of atherosclerosis. The eCB system has several components, including the G-protein-coupled cannabinoid receptors (CB1 and CB2); their endogenous ligands, 2-arachidonoylglycerol (2-AG) and anandamide (AEA); and biosynthetic enzymes that produce and degrading these compounds. CB2 signaling has been shown to upregulate immunoprotective and anti-oxidative pathways, whereas CB1 signaling has opposite effects. We hypothesized a mechanistic link between scavenger receptor activation and Nox activity, which leads to enhanced 2-AG biosynthesis via a signaling pathway that activates diacylglycerol lipase beta (DAGLB). Activation of CB2-mediated signaling by enhanced “eCB tone” can potentially reduce oxidative stress in macrophages. The released 2-AG is subsequently catabolized hydrolytic enzymes, leading to enhanced 2-AGbiosynthesis via activated DAGLB. We first proved that macrophage treated with oxLDL can activate Nox and increase reactive oxygen species production. We used human and mouse macrophages to demonstrate cause and effect. Secondly, we demonstrated that increased levels of superoxide causes enhanced 2-AG biosynthesis within the macrophage, and that upregulation in eCB production is an adaptive response to oxidative stress. Finally, we identified and quantified the serine hydrolases found in smooth muscle cells (SMCs) using an activity-based protein profiling (ABPP)-MudPIT approach that our laboratory has previously done using human macrophages. Additionally, the catabolism of 2-AG by primary SMCs was explored to demonstrate SMCs can hydrolyze 2-AG to its metabolites arachidonic acid and glycerol by the known hydrolytic enzymes. We demonstrated that enhancing endocannabinoid tone within the vessel wall is a valuable strategy to reduce the occurrence of inflammation that leads to atherosclerosis.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24128号 / 医博第4868号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 秀一, 教授 杉田 昌彦, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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