Dissertationen zum Thema „Atypical Chemokine Receptors“

The immune system relies on the correct spatial and temporal positioning of cells in order to function; cells need to be able to move throughout the circulatory system to survey for pathogens, to migrate from their resident sites in tissues when they sense infection or injury to alert other cells, or to migrate to the site of damage or infection to help mobilise a response. These functions often involve chemokines, small cytokines that signal through chemokine receptors, which are G-protein coupled receptors expressed on the cell membrane. Different chemokines are regulated differentially and can be associated with certain tissues or developmental processes, meaning the suite of receptors expressed by each cell type determines which tissues it is capable of entering, and the precise location it takes up once inside the tissue. Atypical chemokine receptors are a class of chemokine receptors that do not initiate the downstream signalling pathways typical of a G-protein coupled receptor, as they do not recruit intracellular G-proteins, and generally don't induce cell migration. Instead, these receptors are thought to function mainly as chemokine scavenging receptors, internalising and destroying their ligands before rapidly recycling to the cell surface. In this way, the levels of chemokines in the body are prevented from becoming oversaturated thus dampening the ability of cells to respond to signals. ACKR3 and ACKR4 are two atypical chemokine receptors that are expressed on endothelial cells and keratinocytes in the skin. Here, I have studied their expression on cultured human lymphatic endothelial cells and keratinocytes, and modulation in response to immune stimuli on these cells using a combination of qPCR and immunofluorescent staining. These strategies revealed that ACKR3 and ACKR4 are expressed on cultured LECs and keratinocytes and may be differentially regulated by both cell types in response to inflammatory stimuli including bacterial (LPS) and viral (Unmethylated CpG DNA) signatures. Although chemokine scavenging activity could not be detected on these cells, these findings suggested a role for ACKRs 3 and 4 in the inflammatory response. Further experiments in vivo explored the expression and modulation of ACKR3/CXCR4 and ACKR4 on epithelial cells including lymphatic endothelial cells, keratinocytes and vascular endothelial cells in the spleen. Flow cytometry was used to examine tissues both at rest and after inflammation (Aldara-mediated psoriasis model, or TPA-mediated contact hypersensitivity model) and investigate the regulation of ACKR3/CXCR4 or ACKR4 in response to these stimuli. Key findings included the strong overlap and differential regulation of ACKR3 and CXCR4 in response to TPA in the infundibulum subset of keratinocytes. Additionally, lymph nodes of Ackr4-/- mice were significantly enlarged after repeated treatments with Aldara. This appeared to be due to CCL19 dysregulation, but adoptive transfer suggested that there was no defect in leukocyte homing in these mice. This suggested an as yet undetermined response to enhanced CCL19 bioavailability that did not prevent the correct migration of leukocytes to secondary lymphoid organs. Overall, these experiments suggested that ACKR3 and ACKR4 are modulated in response to several inflammatory stimuli both in vivo and in vitro, and that the modulation of homeostatic chemokines can play a role in the response to inflammatory events. This was particularly important in the context of skin inflammation, where inflammatory chemokines, CXCR4 and ACKR2 have all been implicated in severity and duration of inflammatory events, but few studies have yet described the potential contributions of ACKR3 or ACKR4.

Les thérapies anti-cancéreuses, notamment l'immunothérapie, ont fait des progrès considérables ces dernières années ; cependant, seul un petit nombre de patients en tire un bénéfice clinique important et durable. Cela s'explique en partie par l'échec des cellules immunitaires cytotoxiques à infiltrer le microenvironnement des tumeurs. L'infiltration immunitaire dépend notamment du réseau de chimiokines, régulé en partie par les ACKRs. Le but de ce projet de thèse était d'étudier les mécanismes impliqués dans la régulation de ACKR2, qui est impliqué dans la régulation du réseau de chimiokines pro-inflammatoires. Des données in silico ont montré que les promoteurs murins et humains de ACKR2 contiennent des éléments de réponse à l'hypoxie. In vitro, les cellules de cancer colorectal, de mélanome et de cancer du sein hypoxiques surexprimaient ACKR2, ce qui n'était plus le cas pour les cellules présentant une délétion de HIF-1α. In vivo, une diminution de ACKR2 était également observée dans les tumeurs délétées en HIF 1α et associée à une hausse de CCL5 et de l'infiltration immunitaire. L'immunoprécipitation de chromatine a montré que HIF-1α se lie directement sur les motifs HRE du promoteur de ACKR2 dans les cellules de mélanome
Anti-cancer therapies, particularly immunotherapy, have made considerable progress in recent years; however, only a small number of patients derive significant and lasting clinical benefit. The incapacity of cytotoxic immune cells to infiltrate the tumor microenvironment explains in part this phenomenon. Immune infiltration depends in particular on the chemokine network, regulated in part by ACKRs. The aim of this thesis was to study the mechanisms involved in the regulation of ACKR2, which regulates the pro inflammatory chemokine network. In silico data showed that both murine and human ACKR2 promoters contain hypoxia response elements. In vitro, hypoxic colorectal, melanoma and breast cancer cells overexpressed ACKR2, which was no longer the case for cells with a deletion of HIF-1α. In vivo, ACKR2 expression was also decreased in HIF-1α-deleted tumors and associated with increased CCL5 and immune infiltration. Chromatin immunoprecipitation showed HIF-1α directly binds onto the HRE motifs of ACKR2 promoter in melanoma cells

The atypical chemokine receptor-2 (ACKR2) is a chemokine decoy receptor that recognises pro-inflammatory CC chemokines. Many studies showed up-regulated inflammation and delayed resolution of inflammatory responses in ACKR2-/- mice. Furthermore, in the absence of ACKR2, lymphatic endothelial cells (LEC) fail to regulate the expression of pro-inflammatory CC chemokines leading to the excessive peri-lymphatic accumulation of leukocytes. As a result, the migration of antigen presenting cells (APC) through lymphatic vessels may be impaired due to lymphatic congestion. In addition, ACKR2 was shown to regulate lymphatic vessel density in the embryonic skin by regulating the proximity of pro-lymphangiogenic macrophages to LEC. Therefore, to address the role of ACKR2 and its significance in 1) APC migration and 2) inflammation-associated lymphangiogenesis, three models of ocular inflammation were used in this work, experimental autoimmune uveoretinitis (EAU), corneal graft rejection and herpes simplex keratitis (HSK). With regard to APC migration, in both EAU and HSK models, this process was fine-tuned to the level of disease in that migration was significantly compromised in ACKR2-/- mice during severe inflammation, but not under mild inflammatory conditions. Furthermore, while the severity of EAU was associated with the migration of APC, this was not so in HSK. In order to study lymphangiogenesis, the transparent avascular cornea provides a good substrate and corneal lymphangiogenesis was studied using both corneal graft model and HSK model. I found that lymphatic vessel density was increased in ACKR2-/- mice compared to wild type mice in corneal graft induced lymphangiogenesis (macrophage mediated), but not altered during early stages of HSK associated lymphangiogenesis (non-macrophage mediated). These findings confirmed that ACKR2 indirectly regulates the process of lymphangiogenesis in a macrophage dependent manner. Although the severity of HSK correlated with the level of lymphangiogenesis, this does not seem to correlate with viral load but rather associated with inflammatory infiltrations in the cornea.

The ability to clear infections and repair injury is dependent on the coordinated migration of immune cells, or leukocytes. These cells can directly destroy invading pathogens and also produce a variety of bioactive factors that promote pathogen clearance. Interactions between immune cells occur both at the site of inflammation and in specialised lymphoid organs throughout the body. The efficiency and specificity of these interactions relies on the production of a family of molecules called chemotactic cytokines, or chemokines, that drive leukocyte migration. Cells express specific profiles of chemokine receptors to ensure they are directed to the appropriate location to exert their immunological function. The field of chemokine biology, already complex, has been further complicated by the discovery of a subfamily of receptors, the atypical chemokine receptors. These molecules lack the ability to couple to signal transduction pathways used by the other chemokine receptors, and are proposed to act as chemokine scavengers or transport molecules. The atypical chemokine receptor CCX-CKR was discovered more than a decade ago but its function in vivo remains unclear. At the beginning of my project, information about this molecule was very limited. The murine receptor binds the CC chemokines CCL19, CCL21 and CCL25, which have well-characterised and critical roles in the development and homeostasis of the immune system as well as in the immune response to infection. Thus, identification of this new receptor, which unlike classical receptors does not induce cell migration in response to ligand binding, presented some exciting possibilities as to how these processes might be regulated in vivo. Reports describing the pattern of expression of CCX-CKR have thus far provided only limited and sometimes contradictory information. Additionally, while in vitro studies from our lab have provided some important clues as to the potential role of the receptor, published in vivo studies were, at the time of commencing this work, limited to one report describing an unvalidated EGFP reporter knock-in transgenic mouse and a conflicting online resource detailing data generated using a LacZ reporter mouse. To understand the true function of this molecule, it is critical to know where it is expressed in vivo and to explore its function on these cells. In this project I set out with the aim of identifying murine tissues and cells expressing CCX-CKR, as well as examining its potential as an in vivo scavenger of chemokine. Related to this, I hoped to uncover any impact of deletion of CCX-CKR on lymphoid tissue cellularity and/or function, both in resting and inflamed conditions. In chapter 3, I present data that identify lymphoid tissues and “barrier” tissues as sites of robustly detectable CCX-CKR mRNA expression. I describe how I have established a novel fluorescent chemokine tetramer-based protocol for the detection of CCL19 receptors, with emphasis on the application of this protocol to identify CCX-CKR activity on specific cell subsets. Using this method, I present evidence that some CD11b+ CD11c+ myeloid subsets in the inguinal lymph node exhibit CCX-CKR dependent internalisation of chemokine. I also describe attempts to fractionate tissues to identify cell populations responsible for the detected whole-tissue expression of CCX-CKR mRNA. The results described in chapter 4 provide support for the hypothesis that CCX-CKR regulates levels of its ligands in vivo, with alterations in chemokine levels in serum and inguinal lymph nodes in the absence of CCX-CKR. I also present evidence demonstrating that deletion of the receptor can influence mRNA levels of the related receptor CCR7. Following on from this, chapter 5 details my analysis of the impact of CCX-CKR on the cellularity of various lymphoid compartments. I present evidence that CCX-CKR influences lymphocyte populations in the peritoneal cavity, with both innate-like and conventional lymphocytes significantly overrepresented in this compartment. The cellularity of the inguinal lymph node, but not the spleen, is subtly altered by deletion of the receptor. Splenic leukocyte cellularity is not affected, either in number or in localisation. In chapter 6, I turn my attention to the possible role of CCX-CKR during the inflammatory response by examining various experimental parameters during a short-term model of induced cutaneous inflammation. This study shows that CCX-CKR deletion alters the cellularity of the myeloid compartment in the draining lymph node and again highlights myeloid subsets as displaying CCX-CKR dependent chemokine internalisation. Finally, I present preliminary data suggesting a protective effect of CCX-CKR deletion during a long-term model of inflammation-driven tumorigenesis. Taken together, my data provide tentative support for the theory that CCX-CKR acts as a chemokine scavenger in vivo. They further indicate that CCX-CKR is involved in regulating cellularity of various lymphoid compartments both at rest and during induced inflammation. In chapter 7 I discuss in detail the implications of my findings in the context of work published since my project began, and highlight growing evidence to suggest a role for CCX-CKR in regulating immune function.

Infiltration of inflammatory cells into the tissue during the inflammatory response is beneficial to the host. Chemokines and their receptors are instrumental in this process by influencing the migration and behaviour of leukocytes in the tissue. However, prolonged inflammation is associated with many diseases. In recent years, a family of atypical receptors have emerged which do not seem to signal. One of these receptors, D6, is able to internalise and degrade 12 pro-inflammatory CC chemokines and has a role in the resolution of the inflammatory response. Here, using a murine transgenic approach, the potential therapeutic role of D6 in suppressing cutaneous inflammation in vivo has been investigated, using a well-characterised model of skin inflammation. In addition, expression of D6 in a range of inflammatory disorders has also been characterised. Transgenic mice were generated (K14D6), using an epidermis-specific transgene, in which expression of the D6 transgene was driven by the human keratin 14 promoter in epidermal keratinocytes. K14D6 mice were validated and we have shown that D6 is expressed in K14D6 but not in wild-type epidermal keratinocytes. The K14D6 transgene was shown to be functional as only K14D6 keratinocytes were able to bind CCL2 and progressively deplete extracellular CCL3. K14D6 mice can dampen down cutaneous inflammation in response to a topical application of TPA. In addition, K14D6 mice displayed reduced infiltration of epidermal T cells and mast cells compared to wild-type mice. Using a microarray approach, we examined the transcriptional consequences of non-ligated D6 and after ligand binding in primary murine keratinocytes from K14D6 and wild-type mice. Although limited conclusions could be made from the microarray data, our results suggest the possibility that non-ligated D6 in murine keratinocytes may have a negative impact on the transcription of some genes, such as chemokines. In a previous study, D6 null mice displayed a human psoriasis-like pathology after chemical induced skin inflammation, suggesting a possible involvement of D6-dysfunction as a contributing factor in the pathogenesis of psoriasis. We have investigated the possible correlation between D6 expression levels and cutaneous disease development. Analysis of skin biopsies revealed that D6 mRNA levels were 8-fold higher in uninvolved psoriatic skin compared to matching psoriatic lesional skin, atopic dermatitis and control skin. In PBMCs, there was no significant difference in D6 mRNA expression in psoriasis patients compared to control. A preliminary study examining surface D6 expression on leukocytes from control and rheumatoid arthritis patients revealed enhanced D6 expression on B cells and myeloid DCs. In this study, we have shown for the first time that increased expression of D6 in vivo can limit cutaneous inflammation, therefore providing a rationale for exploring the therapeutic potential of D6 in human inflammatory diseases. In addition, we provide evidence that D6 expression is dysregulated in inflammatory disorders further suggesting an involvement of this receptor in the pathogenesis of these diseases.

D6 is an atypical chemokine receptor related to CCR1-5 that binds to many inflammatory CC chemokines. Experiments using transfected cell lines have shown that upon binding to a chemokine ligand D6 does not trigger cellular signalling pathways, but rather acts to scavenge the bound ligand. It achieves this by constitutively travelling to and from the cell surface via early and recycling endosomes, internalising chemokines bound when it is at the cell surface. Over time, D6 removes a large amount of ligands from the extracellular compartment. In vivo, this scavenging activity is thought to regulate the level of CC chemokines, and thus controls inflammation locally and systemically. Lack of D6 has been shown to result in elevated amounts of bioavailable chemokines, and is associated with over exuberant inflammatory responses. In human, D6 mRNA and protein is highly expressed in trophoblast-derived gestational tissues. The expression of D6 mRNA in the placenta is by far the highest, compared to other solid tissues being studied. The importance of D6 in protecting the offspring has been demonstrated in animals. In pigs, a defect in D6 expression was discovered in placental attachment sites in endometrium from arresting fetuses. In mice, lack of D6 results in an increase in fetal loss after challenge with lipopolysaccharide (LPS) or antiphospolipid autoantibodies (aPL), and an increase in the number of abnormal pups when mouse embryos are transferred into fully allogeneic pseudo-pregnant female recipients. In view of these results suggesting a critical role for D6 in placental mediated complications, the expression and molecular function of D6 in primary human trophoblast cells were studied, as to date in vitro human studies have utilised the choriocarcinoma cell line BeWo or immortalised cell lines engineered to over-express exogenous D6. Secondly the impact of D6 deficiency on placental structure, chemokine expression and leukocyte abundance in mice was examined. Chapter 3 presents the results of experiments on primary human trophoblasts. Protocols for routine primary trophoblast isolation, purification and culture from fresh term placentas were optimised in our laboratory. D6 mRNA was detected in these primary cells. Using Western blotting, immunofluorescence and flow cytometry, D6 was shown to be present predominantly in the intracellular vesicles of the cells. Competition chemokine uptake assays, analysed by flow cytometry, showed that CCL2 was internalised by trophoblasts using D6. Competitive chemokine scavenging assays, analysed by quantitative Western blot, confirmed that D6 was functioning as a chemokine scavenger on primary human trophoblasts and that it progressively removed substantial quantities of chemokine from medium bathing the cells. This is the first set of experiments that confirms D6 is present, and functioning as a chemokine scavenger in primary human cells. Chapter 4 contains the results from the mouse experiments. Even in an unchallenged environment it was shown that, on the DBA-1 genetic background, D6 deficiency in the mother and pups leads to higher rates of stillbirth and neonatal deaths, resulting in a reduction in the number of pups weaned per litter than their WT counterparts. By gestational age E14, pup weight was significantly smaller in the D6 KO mice. Using stereological techniques, the placenta of the D6 KO mice at this gestation was found to have a smaller labyrinthine zone. The volume of the labyrinthine zone was positively correlated with pup/placenta ratio. These phenotypes could be due to a maternal or fetal effect of D6 deficiency. To ascertain the answer to this question, the experiment at E14 was extended by breeding DBA-1 females heterozygous for the deleted D6 allele (D6 HET) with D6 deficient (D6 KO) males. In this model the phenotypes of D6 KO pups and placentas could be compared with their D6 HET siblings that developed in a mother expressing some D6 (i.e. D6 HET). Although there were no differences in pup weight, placental weight and pup/placenta ratio between these two groups, stereology revealed a decrease in labyrinthine zone volume fraction in the D6 KO placentas in comparison to their D6 HET siblings. The observed fetal compromise and placental defect at E14 was not apparent at the later gestational age of E18. Luminex multiplex protein assay showed an elevated level of circulating chemokine CCL2 in the serum of D6 KO pregnant mice in comparison to their WT counterpart, so loss of chemokine regulation could be responsible for the defects observed in D6 deficient placentas. In summary, D6 deficiency results in an increase in perinatal death, a fundamental defect in placental formation (reduced labyrinthine zone) and dysregulation of circulating chemokine levels. Chapter 5 discusses the mechanisms of D6 in regulating placental formation and reproductive outcome and the novel insights that this work provided into placental D6 function. It also describes the design of future experiments to reveal the precise role of D6 in chemokine regulation and cell signalling in reproductive immunology, and discusses how D6 might contribute to pregnancy outcome in humans.

Thymus colonisation and thymocyte positioning are mediated by interactions involving CCR7 and CCR9 and their respective ligands CCL19/CCL21 and CCL25. These chemokines also interact with the atypical receptor CCRL1, which is expressed in the thymus and has recently been reported to play an important role in normal abT-cell development. Our study has mapped CCRL1 expression within the adult and embryonic thymus, and shows that CCRL1 is expressed within the thymic cortex, at the subcapsular zone, and surrounding vessels at the corticomedullary junction. We have used flow cytometry to show CCRL1 expression predominantly by cortical thymic epithelial cells, but also by a small population of medullary thymic epithelial cells and by a subset of mesenchymal cells. We show, using CCRL1 deficient mice, that CCRL1 suppresses thymocyte progenitor entry into the thymus, and influences the intrathymic positioning of double negative thymocytes. Nevertheless, we have shown that CCRL1-/- mice have no major perturbations in T- cell populations at different stages of thymic differentiation and development. Overall, this study characterises the expression of CCRL1 in key thymic microenvironments, but argues against a major role for CCRL1 in normal thymus development and function.

Chemokines constitute a family of almost 50 small secreted cytokines, recognized by about 20 different 7TM spanning G protein coupled receptors (GPCRs), that activating pertussis toxin sensitive G proteins induce cell migration. These receptor are abundantly expressed by leukocytes and, controlling cell migration, they dictate leukocyte positioning during homeostatic patrolling within peripheral tissues, their maintenance in bone marrow during maturation and in addition mediate their recruitment to inflamed tissues. Upon inflammation in fact a number of chemokines are produced or activated by inflammatory mediators and diffuse within the tissue, generating a chemical gradient along which leukocytes migrate to reach the center of inflammation to contain and remove the insulting factor. This system needs an extremely tight control, since its dysregulation has been demonstrated to be at the basis of different inflammatory diseases, auto-immunity and has also been linked to cancer development. In particular, in this thesis we focused our attention on two regulatory system: post-translational modifications of chemokines, mediated by enzymes specifically released upon inflammation also by immune cells, and on the activity of atypical chemokine receptors, a subfamily of chemokine receptors that despite high structural homology and similar binding properties compared to conventional chemokine receptors, are unable to drive chemotaxis but act instead as key regulators of the chemokine system activity. In detail we looked at the ability of these regulatory mechanisms to modulate chemokine signaling properties generating a biased signaling, an emerging feature of GPCR pharmacology that describes the ability of a given receptor to elicit different or even opposite functional activities depending on the ability of different agonists to stabilize different receptor’s active structural conformation, resulting in different phenotypes mediated by the same receptor. In the chemokine system biased signaling has already been described to occur on different receptors upon binding of their different ligands, therefore during our investigation on chemokine regulatory system signaling we maintained our focus on the ability of these systems to bias chemokine signaling properties in order to better understand how this regulation occurs. To this point we assessed the ability of differently post-translationally modified chemokines to elicit signaling activities on different receptors by measuring in HEK293 cells their potential in inhibiting adenylyl cyclase, a proximal downstream signal of Gα inhibitory proteins activation, and in inducing β-arrestin recruitment to the receptors in energy transfer-based assays. We also compared signaling properties of an atypical chemokine receptor to the ones elicited by a conventional receptor analyzing the phosphoproteome modifications occurring constitutively and after stimulation with the same agonist. Our results indicate that regulation of CXCL5 and CXCL8 chemokine activity by post-translational modifications is more prone to regulate chemokine activity modifying chemokine potency, rather than generating a bias in their signaling properties. Truncation of chemokine NH2-terminus increases both CXCL5 and CXCL8 activity, while citrullination of the most NH2-terminal arginine results in opposite effects on the two agonists since on CXCL8 increases chemokine potency, while it reduces CXCL5 activity. We investigated the properties of ACKR2 in recruiting β-arrestins, demonstrating that this receptor is able to associate both β-arrestin 1 and 2 in basal conditions while upon agonist stimulation preferentially increases its association with β-arrestin 1, resulting in a completely different agonist-induced outcome of proteome phosphorylation, compared to CCR5, in terms of kinetics, protein phosphorylation modifications, biological function of the regulated proteins and signal mediators activated. Taken together, these results indicate that chemokine system regulation is based not only on chemokine post-translational modifications that modulate chemokine potency, but also on the activity of structurally biased atypical chemokine receptors, as in the case of ACKR2 that interacts with different effectors and kinetics to generate distinct functional outcomes, compared to the conventional chemokine receptor CCR5. In this thesis it has also been attempted to translate the investigation of chemokine signaling to a clinical intervention for inflammatory diseases. We assessed the modulation of CXCR1 signaling activity exerted by Reparixin, a leukocyte migration inhibitor that blocks cell recruitment to inflamed tissues, that binds to its target receptors in an allosteric binding site, without inhibiting chemokine binding to the receptor. In our assays performed on HEK293 cells we could not detect any inhibition of the signaling pathways assayed, possibly indicating that HEK293 cells are not the best model to assay the activity of this molecule, with the need to assess in this cellular model drug inhibitory activity on read-outs already evaluated in literature on other cell types. In conclusion, we can say that observations described in this thesis allow to better understand chemokine system regulation, that occurs by biased signaling activity in the case of atypical chemokine receptors that by selected interaction with signaling mediators induce opposite biological outcomes compared to conventional chemokine receptors, while post-translational modifications regulate chemokines activity modulating their potency, rather than biasing their signaling properties.

Psoriasis is a common, debilitating systemic inflammatory disorder that is characterised by sharply demarcated, thick, erythematous scaly skin plaques. Such plaques commonly appear on skin that is subjected to repeated tensile trauma, such as elbows, knees and flexures. The mechanism by which these inflammatory lesions are spatially restricted is not known and yet knowledge of this could be of critical importance for our understanding of this disease. Chemokines are the principal regulators of leukocyte migration and play a critical role in the initiation and maintenance of inflammation. The atypical chemokine receptor ACKR2 (formerly D6) binds inflammatory CC-chemokines, but does not signal upon ligand binding; instead ACKR2 internalises and helps degrade such chemokines, after which it continues to cycle back to the cell surface. ACKR2 acts, through this mechanism, as a high-capacity scavenger of chemokines, and plays an important role in regulating inflammation. It is known that ACKR2 expression is high in unaffected skin in patients with psoriasis (remote from inflammatory plaques) and concurrently deficient in the plaques themselves. Additionally, human studies have shown that simple skin trauma in psoriasis patients causes a reduction in cutaneous ACKR2 expression at the site of trauma. However, the functional significance and the molecular mechanism by which it occurs are not understood. This thesis explored the role of ACKR2 in the spatial restriction of psoriasiform inflammation and the molecular mechanisms for its differential regulation. Through the use of disease relevant mouse models, primary human cell cultures and novel cell migration assays, the results presented here show that localised psoriasiform inflammation upregulates ACKR2 in remote tissues through the systemic release of cytokines. This remotely upregulated ACKR2 expression protects tissues from the further spread of inflammation. This protective effect is mediated by stromally expressed ACKR2 that acts to control inflammatory T-cell positioning within the skin. Tensile trauma of keratinocytes however, acted to reduce ACKR2 expression in the context of inflammation, which in turn provides a novel mechanism for the well-characterised phenomenon that occurs in psoriasis (and a range of skin condition) termed ‘koebnerisation’. Koebnerisation refers to the phenomenon by which relatively simple skin trauma induces the development of disease-specific skin lesions. Furthermore, this thesis defines novel disease-relevant regulators of ACKR2 expression. In silico analyses identified psoriasis-associated microRNAs that bound to the 3’-UTR of ACKR2, and reduced its expression at transcriptional and protein level. Importantly, trauma of keratinocytes induced ACKR2 downregulation concurrent with a substantial and significant increase in the expression of the identified ACKR2 targeting microRNAs. Together, this thesis defines a novel mechanism by which ACKR2-mediated regulation of chemokine function, cutaneous trauma, microRNAs and systemic cytokines, co-ordinately modulate the predisposition of remote tissue sites to the development of new lesions. Importantly, the results presented here have profound implications for how spatial restriction is imposed on inflammation. The data also highlight therapeutic ACKR2 induction as a plausible novel strategy for the limitation and treatment of psoriasiform- and potentially other forms of inflammation.

The chemokine receptor CXCR3 and its agonists CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC are involved in a variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. CXCL11 has also been reported to bind to an additional receptor, namely CXCR7, which also interacts with CXCL12. Two alternatively spliced variants of the human CXCR3 receptor have been described, namely CXCR3-B and CXCR3-alt. The human CXCR3-B has been found to bind CXCL9, CXCL10, CXCL11 as well as an additional agonist CXCL4/PF4. In contrast, CXCR3-alt only binds CXCL11. This work demonstrates that CXCL4 like the original CXCR3 agonists is capable of inducing biochemical signalling, namely intra-cellular calcium elevation, and activation of p44/p42 MAPK and PI3K/Akt pathways in activated human T lymphocytes. Phosphorylation of p44/p42 MAPK and Akt was inhibited by pertussis toxin, suggesting coupling to Gi protein. In contrast CXCR3 antagonists blocked CXCR3 agonists but not CXCL4-mediated responses. Surprisingly, stimulation of T cells with CXCL4 failed to elicit migratory responses of these cells and did not lead to loss of surface CXCR3 expression. Collectively our evidence shows that although CXCL4 is coupled to downstream biochemical machinery, its function in T cells is distinct from the function of CXCR3 agonists. The work presented in this thesis also indicates that despite considerably lower surface expression in comparison to the full length CXCR3, CXCR3-B and CXCR3-alt transduce biochemical signals in response to CXCL11 in transfected cells. According to previous reports the role of CXCR7 in signalling and chemotaxis in T cells could not be detected. In T cells and transfected cells system CXCR7 was localised at the plasma membrane and was efficiently internalized in response to CXCL11 and CXCL12. Studies of the involvement of methylation in T cell chemotaxis suggest that this modification may be required in this process as it was partially inhibited by methylation inhibitor- MTA. Moreover T cell co-stimulation caused increased levels of arginine mono-methylated proteins suggesting the importance of methylation in T lymphocyte signalling.

Chemokines promote leukocyte migration through the activation of dedicated G-protein coupled receptors. Beyond conventional chemokine receptors, which directly induce cell migration through heterotrimeric Gαi-mediated signalling events, a set of atypical chemokine receptors (ACKRs) have been described. ACKRs do not activate Gαi-mediated signalling activity, but they are mainly involved in shaping the chemokine gradient. The best characterized member of this family is ACKR2. ACKR2, previously referred to as D6, is a scavenger receptor that binds with high affinity to 13 inflammatory CC chemokines. The scavenging activity of ACKR2 relies on its intracellular traffic properties. Under homeostatic conditions, ACKR2 is mainly localized in intracellular stores associated with both early Rab4/5-positive and recycling Rab11-positive endosomes. At increasing levels of chemokines, ACKR2 increases plasma membrane abundance through an acceleration in the rate of Rab11-depedent recycling pathway, in order to optimize its chemokine scavenging activity. Here, I demonstrated that the intracellular distribution of ACKR2 is maintained by cytoskeletal dynamics. After chemokine engagement, ACKR2 activate a G-protein-independent and β-arrestin-dependent Rac1-PAK1-LIMK1 signalling cascade to finely regulate the actin cytoskeletal and the microtubules network reorganization, to promote receptor up-regulation and scavenging function. ACKR2 is able to recruit and associates both β-arrestins in basal condition, at membrane and intracellular levels, but only β-arrestin1 is recruited after active ligand stimulation, in order to promote a β-arrestin1-dependent signalling pathway, required for supporting the myosin Vb-dependent ACKR2 up-regulation and scavenging properties.

Chemokines and chemokine receptors are key mediators of inflammation and important regulators of leukocyte migration in homeostatic conditions as well as during infection and cancer. The atypical receptor ACKR2 is a scavenger receptor for many inflammatory CC chemokines, it is expressed either by non-hematopoietic cells or by hematopoietic cells, and it has been shown to prevent the development of exacerbated inflammatory reactions. In an effort to understand the contribution of this receptor in the regulation of myeloid cell mobilization and myeloid cell effector functions, we investigated the role of ACKR2 in a murine model of myeloid cell mobilization, and in a model of experimental metastasis. The deficiency of ACKR2 was associated with increased mobilization of monocytes and neutrophils from the bone marrow (BM) and with increased number of monocytes confined to BM sinusoids compared to Wild-type (WT) mice. BM chimera experiments showed that the increased mobilization was due to the absence of ACKR2 in the hematopoietic compartment. The analysis of hematopoietic progenitor cells (HPCs) revealed that ACKR2 is expressed by Lin−Sca-1+c-Kit+ cells (LSK) to faint thereafter in more mature myeloid progenitor cells (MPCs) in contrast with the canonical chemokine receptor CCR2. Moreover, HPCs from Ackr2-/- mice expressed higher levels of CCR1, CCR2 and CCR5, but not of CXCR4 and they had higher differentiation rate compared to ACKR2 sufficient LSK. Although neutrophils express low levels of ACKR2 compared to LSK, we found that neutrophils from Ackr2 deficient mice, as well as their HPCs, expressed higher level of CC chemokine receptors and exhibited a more activated phenotype compared to WT. Furthermore, neutrophil depletion and neutrophil adoptive transfer experiments demonstrated that only Ackr2 deficient neutrophils were sufficient to control the metastatic seeding of B16 melanoma cells into the lung. To enhance the metastatic protection observed in Ackr2-/- mice, we treated WT and Ackr2-/- tumor bearing mice with AMD3100, the competitive inhibitor of CXCR4, which is known to induce a rapid neutrophil mobilization from the BM. However, AMD3100 treatment did not further improve the metastatic protection in Ackr2-/- mice, whereas decreased the number of metastases in WT mice. Finally, by using the human promyelocytic cell line HL-60, we demonstrated that ACKR2 directly exerted a negative regulation of CC chemokine receptor expression and cell differentiation. Indeed, HL-60, when transfected with a vector overexpressing ACKR2, had decreased transcript levels of CCR2 and CD11b. These data suggest the ACKR2 is involved in the regulation of chemokine availability and leukocyte recruitment. Moreover, ACKR2 directly controls HPC differentiation, myeloid cell mobilization and their effector function through the inhibition of CC chemokine receptor expression.

The CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3) are seven transmembrane receptors that are involved in numerous pathologies, including several types of cancers. Both receptors bind the same chemokine, CXCL12, leading to significantly different outcomes. While CXCR4 activation generally leads to canonical GPCR signaling, involving Gi proteins and β‐arrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via β‐arrestin‐dependent signaling pathways. Understanding the dynamics and kinetics of their activation in response to their ligands is of importance to understand how signaling proceeds via these two receptors. In this thesis, different Förster resonance energy transfer (FRET)‐based approaches have been combined to individually investigate the early events of their signaling cascades. In order to investigate receptor activation, intramolecular FRET sensors for CXCR4 and ACKR3 were developed by using the pair of fluorophores cyan fluorescence protein and fluorescence arsenical hairpin binder. The sensors, which exhibited similar functional properties to their wild‐type counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RET‐based receptor sensors in the field of chemokine receptors. Additional FRET‐based settings were also established to investigate the coupling of receptors with G proteins, rearrangements within dimers, as well as G protein activation. On one hand, CXCR4 showed a complex activation mechanism in response to CXCL12 that involved rearrangements in the transmembrane domain of the receptor followed by rearrangements between the receptor and the G protein as well as rearrangements between CXCR4 protomers, suggesting a role of homodimers in the activation course of this receptor. This was followed by a prolonged activation of Gi proteins, but not Gq activation, via the axis CXCL12/CXCR4. In contrast, the structural rearrangements at each step of the signaling cascade in response to macrophage migration inhibitory factor (MIF) were dynamically and kinetically different and no Gi protein activation via this axis was detected. These findings suggest distinct mechanisms of action of CXCL12 and MIF on CXCR4 and provide evidence for a new type of sequential signaling events of a GPCR. Importantly, evidence in this work revealed that CXCR4 exhibits some degree of constitutive activity, a potentially important feature for drug development. On the other hand, by cotransfecting the ACKR3 sensor with K44A dynamin, it was possible to increase its presence in the plasma membrane and measure the ligand‐induced activation of this receptor. Different kinetics of ACKR3 activation were observed in response to CXCL12 and three other agonists by means of using the receptor sensor developed in this thesis, showing that it is a valuable tool to study the activation of this atypical receptor and pharmacologically characterize ligands. No CXCL12‐induced G protein activation via ACKR3 was observed even when the receptor was re-localized to the plasma membrane by means of using the mutant dynamin. Altogether, this thesis work provides the temporal resolution of signaling patterns of two chemokine receptors for the first time as well as valuable tools that can be applied to characterize their activation in response to pharmacologically relevant ligands
Der CXC Chemokin‐Rezeptor 4 (CXCR4) und der atypische Chemokin‐Rezeptor 3 (ACKR3) sind heptatransmembranäre Rezeptoren, die in zahlreichen Krankheitsbildern eine Rolle spielen, wie in einigen Krebsarten. Beide Rezeptoren werden zwar von dem gleichen Chemokin CXCL12 aktiviert, allerdings mit unterschiedlichen Signalweiterleitungsmustern. Die Aktivierung von CXCR4 führt zu kanonischer GPCR Signaltransduktion über Gi‐Proteine und β‐Arrestine. Die Signalweiterleitung des Rezeptors ACKR3 hingegen, welcher hauptsächlich in intrazellulären Vesikeln vorliegt, erfolgt über ß‐Arrestinabhängige Signalwege. Es ist von großer Wichtigkeit die Dynamik und Kinetik dieser beiden Rezeptoren hinsichtlich der Aktivierung durch ihre Liganden und der Signalweiterleitung zu verstehen. In dieser Arbeit wurden verschiedene Förster‐Resonanzenergietransfer (FRET) Anwendungen kombiniert, um die frühen Phasen der Signal‐Kaskade von CXCR4 und ACKR3 zu untersuchen. Zur genaueren Aufklärung der Rezeptoraktivierung wurden intramolekulare FRET‐Sensoren entwickelt, hierzu wurden die Fluorophore Cyan‐fluoreszierendes Protein und engl. fluorescence arsenical hairpin binder verwendet. Die generierten Sensoren zeigten ähnliche funktionelle Eigenschaften wie die unveränderten Rezeptoren. Liganden‐induzierte Änderungen der Rezeptorkonformation können mittels dieser Sensoren beobachtet werden und stellen die ersten RET‐basierten Sensoren auf dem Forschungsgebiet der Chemokin‐Rezeptoren dar. Weitere FRET‐basierte Methoden wurden zur Untersuchung von Interaktionen zwischen Rezeptor und G‐Protein, Neuanordnung von Dimeren, sowie der G‐Protein Aktivierung eingesetzt und für beide Chemokin‐Rezeptoren etabliert. CXCR4 zeigte einen komplexen Aktivierungsmechanismus nach Stimulation durch CXCL12, bei welchem zunächst eine Neuordnung der Rezeptor‐Transmembrandomäne gefolgt von Neuordnungen zwischen Rezeptor und G‐Protein und zuletzt eine Neuordnung zwischen CXCR4 Protomeren erfolgte. Dies impliziert, dass im Aktivierungsprozess des Rezeptors Homodimere eine Rolle spielen. Zudem wurde eine verlängerte Gi ‐Protein Aktivierung gegenüber der Gq‐Protein Aktivierung bei CXCL12 stimuliertem CXCR4 beobachtet. Hingegen zeigte eine Stimulierung mit dem Macrophage Migration Inhibitory Factor (MIF) bei jedem Schritt der frühen Singal‐Kaskade veränderte Dynamiken und Kinetiken im Vergleich zu CXCL12. Darüber hinaus konnte keine Gi ‐Protein Aktivierung festgestellt werden. Dieser Befund zeigt individuelle Mechanismen für MIF und CXCL12 am CXCR4‐Rezeptor und liefert Belege für eine neuer Art von sequenziellen Signalweiterleitungen an GPCRs. Eine wichtige Beobachtung dieser Arbeit für eine potentielle Medikamentenentwicklung ist das CXCR4 ligandenunabhängige Aktivität zeigt. Um die Aktivierung des ACKR3 Sensors messen zu können wurde durch eine Co‐Transfektion mit K44A Dynamin eine höhere Membranständigkeit erreicht. CXCL12 und drei weiteren Agonisten zeigten am hier entwickelten ACKR3‐Sensor unterscheidbare Kinetiken. Mit diesem wertvollen Werkzeug können Liganden an diesem atypischen Rezeptor pharmakologisch charakterisiert werden. Es konnte keine CXCL12‐induzierte G‐Protein Aktivierung gemessen werden, trotz der stärkeren Präsenz an der Plasmamembran mit Hilfe der Dynamin‐Mutante. In Summe liefert diese Arbeit zum ersten Mal eine zeitliche Auflösung von Signalweiterleitungsmustern von zwei Chemokin‐Rezeptoren sowie wertvolle Werkzeuge zur Charakterisierung der frühen Phase der Signal‐Kaskade durch andere pharmakologisch relevanten Liganden

The significance of chemokine receptors CCR7, CCR9 and their ligands CCL19, CCL21, and CCL25 in various types of cancer including mammary carcinoma and melanoma has been highlighted over the last decade. The atypical chemokine receptor CCK-CKR is a high affinity receptor for these chemokine ligands but rather than inducing classical downstream signalling events promoting migration, it instead sequesters and targets its ligands for degradation. Therefore, CCX-CKR has been proposed to regulate chemokine bioavailability in vivo. This putative function of CCX-CKR to regulate the levels of pro-tumourigenic chemokines initially led to the hypothesis that local and systemic regulation of chemokine levels by CCX-CKR influences tumour growth and metastasis in vivo, and ultimately, targeting of CCX-CKR could be an effective cancer therapy. Three broad approaches were taken to investigate the role of CCX-CKR in tumour progression and metastasis including overexpression of the receptor on tumour cells, deletion from the mouse host and receptor expression knockdown in tumour cells. The results revealed that overexpression of CCX-CKR on 4T1.2 mouse mammary carcinoma cells inhibits orthotopic tumour growth. However, this effect could not be correlated with chemokine scavenging in vivo and was not attributed to host adaptive immunity from experiments performed during the course of the current study. On the other hand, overexpression of CCX-CKR on 4T1.2 cells also resulted in enhanced spontaneous metastasis and haematogenous metastasis in vivo. In vitro characterisation of tumourigenicity of 4T1.2 cells revealed that overexpression of CCX-CKR rendered them more invasive, less adherent to the ECM and to each other and more resistant to anoikis. These are established characteristics of cells which have undergone EMT and indeed, CCX-CKR overexpressing cells showed a typical expression pattern of EMT markers. In contrast, when endogenous expression of CCX-CKR is deleted in the mouse host, growth and metastasis of E0771 mammary carcinoma and B16 melanoma are inhibited, which is accompanied by elevated levels of CCX-CKR ligands in tumours and relevant naïve tissues from CCX-CKR-deleted mice. Similarly, shRNA-mediated knockdown of endogenous CCX-CKR from B16 melanoma cells leads to the rejection of primary and secondary tumours. This effect is attributed to elevated levels of CCX-CKR ligands and CCR7⁺ and CCR9⁺ leukocytes in tumour tissues, which resulted in an overall enhancement of the host anti-tumour immune response. Consistent with these observations, growth of CCX-CKR knockdown tumours was comparable to that of control tumours in CCR7-deleted mice indicating host CCR7 dependency of CCX-CKR-mediated rejection of B16 melanoma. Together, findings from this study revealed important insights into the complex role of CCX-CKR in cancer progression and highlights CCX-CKR as a novel target for the development of more effective anti-melanoma therapies and potentially for the treatment of other types of cancer which affect millions of people worldwide.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2012

The significance of chemokines in directing cell migration both during homeostasis and immune responses has been appreciated for some time. However, the mechanisms in place to post-translationally regulate cell migration through chemokine modulation are only recently becoming clear. CCX-CKR is a receptor that can scavenge and degrade the ligands of CCR7 and CCR9, two receptors that are crucial during instruction of T cell development in the thymus and dendritic cell migration for initiation of adaptive immune responses. Within the thymus CCL19, CCL21 and CCL25 direct CCR7- and CCR9-expressing thymocytes through distinct thymic compartments, enabling development of a self-MHC restricted and self-tolerant peripheral T cell repertoire. Yet mechanisms outside of transcriptional control that are involved in thymic chemokine regulation have not been well characterised. The aim of this study was to thoroughly investigate the role of CCX-CKR expression on chemokine regulation in the thymus and thymocyte development. Expression of CCX-CKR was detected primarily in cortical thymic epithelial cells, with modest contributions from other thymic stromal populations. Deletion of CCX-CKR led to thymic architecture alterations, reduced levels of CCL19 and CCL25 and a profound decrease in CCL25 (protein) within the cortex. These alterations in chemokine levels and distribution were associated with several defects in the frequency and localisation of thymocyte precursors. Specifically, in CCX-CKR⁻/⁻ thymi, precursor double-negative 2 (DN2) cells accumulated in the medulla and reduced frequencies of DN3 cells were apparent, coincident with reduced numbers of DN3 cells in the cortex. These observations are likely to be the combined outcome of impaired expansion of cortical thymic epithelial cells and reduced outward migration signals in CCX-CKR⁻/⁻ thymi. Additionally, CCXCKR ⁻/⁻ thymi contain increased numbers of CD4⁺CD8⁺ double-positive, CD4⁺ singlepositive and CD8⁺ single-positive cells. Together, these thymic defects were associated with enhanced incidence of inflammatory pathology resembling Sjögren’s syndrome, characterised by lymphocytic infiltrates in salivary glands and liver of 8-10 month old CCX-CKR⁻/⁻ mice. Previous work has implicated CCX-CKR as an important regulator of CCL19 and CCL21 in vivo and deletion of CCX-CKR led to early onset of experimental autoimmune encephalomyelitis (EAE), a T cell mediated autoimmune disease. CCX-CKR was also implicated in promoting effective induction of adaptive immune responses in the LN as evidenced by abrogated T cell proliferation. An important component of both peripheral tolerance induction and initiation of adaptive immune responses is CCR7-dependent migration of antigen-loaded peripheral dendritic cells and naïve T cells to secondary lymphoid organs where antigen presentation to T cells occurs. The contribution of CCXCKR to these processes was investigated in CCX-CKR⁻/⁻ mice. Short-term homing of CD4⁺ T cells to the lymph nodes of CCX-CKR⁻/⁻ mice was impaired yet homeostatic maintenance of T and B cells remained undisturbed. CD207⁺ skin-derived DCs were significantly less abundant in the lymph nodes of CCX-CKR⁻/⁻ mice during both steadystate and inflammation which was associated with reduced numbers of CD207⁺ dermal dendritic cells and increased levels of CCL21 in the skin. Furthermore, during CFAinduced inflammation, both migratory and lymph node-resident dendritic cell numbers were abrogated in the lymph nodes, but not spleen, of CCX-CKR⁻/⁻ mice. Together, these data identify a novel role for CCX-CKR in maintenance of the thymic cortical compartment that is associated with effective thymocyte development. Moreover, CCX-CKR is required to maintain the homeostatic and inflammatory migration of tolerogenic and activating dendritic cells, respectively, to the lymph nodes. Thus, the combination of thymic and skin/LN associated CCX-CKR establishes optimal conditions for central and peripheral tolerance induction leading to the development of self-tolerant adaptive immune responses.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular & Biomedical Science, 2012