<p>Mast cells are very potent multifunctional effector cells of the immune system normally distributed throughout connective tissues. An accumulation of mast cells has been described in several pathological conditions such as allergic- and autoimmune inflammations and in certain tumours. This necessitates two different processes: 1) Recruitment of mast cell progenitors from peripheral blood; 2) Accretion of mature mast cells at sites of inflammation and tumour areas. Both processes are depending on the local production of chemotactic factors. The aim of this study was to investigate the role of chemokines and their corresponding receptors in mast cell chemotaxis. </p><p>By cloning and mRNA-screening of cord blood derived mast cells several chemokine receptors were found to be expressed. Functional expression was confirmed of chemokine receptors CXCR4, CCR1 and CCR4. CXCL12, the only known ligand for CXCR4, acted as a mast cell chemotaxin and induced migration of progenitor cells with capacity to differentiate into mast cells. Of several ligands known to bind CCR1 and CCR4, only CCL5 induced migration of mast cells. The migration to CCL5 was mediated through both CCR1 and CCR4. In contrast, the ligands to CCR4, CCL17 and CCL22, could inhibit CCL5-induced migration. Expression of CCR1 and CCR4 could also be confirmed on mast cells in lung from asthmatic patients. Furthermore, we could demonstrate that mast cells were attracted by CCL5 produced by tumour cells in Hodgkin´s lymphoma.</p><p>In conclusion, the work in this thesis has identified two chemokines that regulates mast cell migration. This knowledge helps us understand the mechanisms behind homing of mast cell progenitors from the blood into the tissue and the accumulation of mature mast cells at sites of inflammation and tumourigenesis.</p>

Mast cells are very potent multifunctional effector cells of the immune system normally distributed throughout connective tissues. An accumulation of mast cells has been described in several pathological conditions such as allergic- and autoimmune inflammations and in certain tumours. This necessitates two different processes: 1) Recruitment of mast cell progenitors from peripheral blood; 2) Accretion of mature mast cells at sites of inflammation and tumour areas. Both processes are depending on the local production of chemotactic factors. The aim of this study was to investigate the role of chemokines and their corresponding receptors in mast cell chemotaxis. By cloning and mRNA-screening of cord blood derived mast cells several chemokine receptors were found to be expressed. Functional expression was confirmed of chemokine receptors CXCR4, CCR1 and CCR4. CXCL12, the only known ligand for CXCR4, acted as a mast cell chemotaxin and induced migration of progenitor cells with capacity to differentiate into mast cells. Of several ligands known to bind CCR1 and CCR4, only CCL5 induced migration of mast cells. The migration to CCL5 was mediated through both CCR1 and CCR4. In contrast, the ligands to CCR4, CCL17 and CCL22, could inhibit CCL5-induced migration. Expression of CCR1 and CCR4 could also be confirmed on mast cells in lung from asthmatic patients. Furthermore, we could demonstrate that mast cells were attracted by CCL5 produced by tumour cells in Hodgkin´s lymphoma. In conclusion, the work in this thesis has identified two chemokines that regulates mast cell migration. This knowledge helps us understand the mechanisms behind homing of mast cell progenitors from the blood into the tissue and the accumulation of mature mast cells at sites of inflammation and tumourigenesis.

Avec 42 000 nouveaux cas diagnostiqués en 2012, le cancer colorectal (CCR) représente en France le troisième cancer en termes d’incidence. Les métastases, qui surviennent principalement au niveau du foie et des poumons, en constituent la principale cause de décès. Malgré les progrès récents de la chimiothérapie et des agents ciblés, le taux de survie à 5 ans des patients présentant un CCR métastasé reste faible. Aujourd’hui, la résection chirurgicale est le seul traitement curatif, cependant moins de 20% des patients porteurs de métastases sont opérables. Il existe donc un grand nombre de patients présentant un CCR métastasé pour lequel aucun traitement curatif ne peut être proposé. La formation des métastases à partir d’une tumeur primaire résulte d’une longue série d’étapes séquentielles liées les unes aux autres. L’issue de ce processus dépend à la fois des propriétés intrinsèques des cellules tumorales et de la réponse de l’hôte. Il a récemment été montré que les couples chimiokines/récepteurs interviennent dans le contrôle des différentes étapes de la progression tumorale.Le projet de recherche développé au cours de mon travail de thèse avait pour objectif d’utiliser les chimiokines et leurs récepteurs dans de nouvelles stratégies thérapeutiques pour bloquer et/ou éradiquer les métastases hépatiques et pulmonaires des CCRs. Le travail s’est articulé selon deux axes dans lesquels nous avons montré d’une part que, le blocage du récepteur de chimiokines CXCR7 permet de limiter les métastases pulmonaires de CCRs et d’autre part que, le transfert de gène codant CX3CL1 au niveau du foie entraîne une réponse anti-tumorale efficace dans les métastases hépatiques de CCRs. / With 42 000 newly-diagnosed patients in 2012, the colorectal cancer (CRC) represents the third type of cancer in terms of incidence in France. The leading cause of death from CRC is the development of metastases and these metastases will occur mostly within the liver (50% of the patients) and within the lungs (15%). Despite recent progress, notably in the chemotherapies now used and the targeted agents, the rate of 5-years survival for late stage CRC remains low. Nowadays, the surgical resection is the only curative treatment proposed to patients with metastatic CRC, however less than 20% of them have an operable tumour. There is therefore a high number of patients for whom no cure is currently available. A primary tumour’s dissemination to a second organ is the result of a long process made of numerous cross-linked steps. The final outcome of this process depends on the intrinsic properties of tumour cells as well as the host response. Recently, it has been shown that the chemokine-chemokine receptor pairs (initially described as regulating the leukocyte migration) play crucial roles in the various stages involved in tumour progression. The aim of the research project developed during my PhD was to assess the use of the chemokines and their receptors in new therapeutic strategies to block and/or eradicate the hepatic and pulmonary metastases of CRC. Our work has been organized along two main lines of approach. We have shown that the blockage of the CXCR7 chemokine receptor enables the limitation of the CRC metastases within the lungs and that the CX3CL1 gene transfer into the hepatocytes leads to an efficient anti-tumor response in the CRC metastases within the liver.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-10-21T12:20:22Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Adriene Siqueira de Melo_Medicina Tropical_2016_Publicação Digital.pdf: 2979717 bytes, checksum: 57f59f57f32268ca22f79a21050c7978 (MD5) / Made available in DSpace on 2016-10-21T12:20:22Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Adriene Siqueira de Melo_Medicina Tropical_2016_Publicação Digital.pdf: 2979717 bytes, checksum: 57f59f57f32268ca22f79a21050c7978 (MD5) Previous issue date: 2016-02 / CAPES / Por contribuírem nos processos de migração diferencial de células imunes aos tecidos, quimiocinas e seus receptores estão potencialmente implicados na manutenção do infiltrado inflamatório observado na miocardiopatia chagásica crônica. Assim, avaliamos em pacientes com doença de Chagas crônica a associação da produção de quimiocinas e da expressão fenotípica para receptores de quimiocinas por células T CD4+, CD8+ e CD4+CD8+, com a presença e gravidade da manifestação cardíaca. Cinquenta e seis pacientes (Forma indeterminada, IND=20; Forma cardíaca leve, CARD1=20 e Forma cardíaca severa, CARD2=16), foram selecionados no Ambulatório de Doença de Chagas do Pronto-Socorro Cardiológico de Pernambuco/Universidade de Pernambuco. O sangue foi cultivado (1dia/37ºC/5%CO2) na presença de Antígeno Solúvel de Epimastigota (25μg/mL), de Fitohemaglutinina (5μg/mL) e mantendo-se um tubo sem estímulo. O sobrenadante e as células sanguíneas foram analisados por citometria de fluxo e as diferenças consideradas quando p<0,05. O grupo CARD2 apresentou maiores níveis de células T CD8+CCR1+, CD8+CCR3+, CD4+CD8+CCR1+, CD4+CD8+CCR3+, de Intensidade média de fluorescência (MIF) para CCR1 e CCR3, e de produção de CCL5 e CXCL8 que o grupo IND, além de maior produção para CXCL10 que o CARD1. O grupo CARD1 apresentou maiores níveis para células T CD4+CXCR5+, CD4+CCR4+, de MIF para CXCR5 e de produção de CXCL8 que os pacientes IND. É possível que as células e quimiocinas diferencialmente observadas nos indivíduos com a manifestação cardíaca estejam atuando por mecanismos de citotoxidade, oriundos tanto da resposta imune específica ao parasita quanto de possíveis processos autoimunes, para o estabelecimento dos danos tissulares e sintomatologia clínica nestes pacientes. / Contributing to differential migration of immune cells to tissues, chemokines and their receptors are potentially involved in the maintenance of inflammatory infiltrate observed in chronic Chagas' cardiomyopathy. We evaluated in chronic Chagas' disease patients the association of chemokine production and phenotypic expression of chemokine receptors by CD4+, CD8+ and CD4+ CD8+ T cells with the presence and severity of heart involvement. Fifty-six patients (indeterminate form, IND = 20; mild heart shape, CARD1 = 20; and severe heart shape, CARD2 = 16) were selected at Ambulatório de Doença de Chagas, Pronto-Socorro Cardiológico de Pernambuco/Universidade de Pernambuco.The blood was cultured (1day / 37°C/5% CO2) in the presence of epimastigote antigen (25 μg / ml), Phytohaemagglutinin (5μg / ml) and an unstimulated culture was also performed. The supernatant and the blood cells were analyzed by flow cytometry and the differences were considered when p <0.05. The CARD2 group had higher levels of CD8+CCR1, CD8+CCR3+, CD4+CD8+CCR1+ and CD4+CD8+CCR3+ T cells; higher mean intensity of fluorescence (MIF) to CCR1 and CCR3, and higher production of CCL5 and CXCL8 than IND. Furthermore, CARD2 had increased production of CXCL10 than CARD1. The CARD1 group showed higher levels of CD4+CXCR5+ and CD4+CCR4+ T cells, higher MIF for CXCR5 and higher production to CXCL8 than IND. It is possible that cells and chemokines found in such individuals presenting cardiac involvement, are contributing to cytotoxicity mechanisms raised by the specific immune response to the parasite or the possible autoimmunity, in establishing of cardiac tissue injuries with clinical manifestation.

Notre objectif était d’analyser l’implication potentielle des voies associées aux récepteurs de chimiokines CXCR2 et CXCR4 dans le cancer colorectal métastatique au foie. Les niveaux d’expression de CXCR2, CXCR4 et de leurs chimiokines étaient évalués dans les métastases hépatiques de cancers colorectaux dans le but d’étudier leurs corrélations avec la survie globale et la survie sans récidive de patients ayant reçu, ou non, une chimiothérapie néoadjuvante. Des analyses d’expression pour RT-PCR quantitative et immunohistochimie étaient réalisées en utilisant des prélèvements humains de métastases hépatiques de cancers colorectaux. Les niveaux d’expression de CXCR2, CXCR4 et de leurs ligands étaient statistiquement analysés en fonction des traitements par chimiothérapie néoadjuvante administrés ou non, et en fonction du suivi des patients. Des modèles murins de xénogreffes sous-cutanées et orthotopiques intracaecales ont été mis au point et utilisés pour étudier l’expression de CXCR2, CXCR4 et CXCL7 en relation avec le traitement des souris par chimiothérapie.Nous avons montré que la surexpression de CXCR2 et CXCL7 était corrélée à de plus courtes survies globales et sans récidive de nos patients. En analyse multivariée, l’expression de CXCR2 et de CXCL7 étaient des facteurs indépendants de survie globale et sans récidive. La chimiothérapie néoadjuvante augmentait significativement l’expression de CXCR2, et de CXCL7 de façon proche de la significativité. Les résultats de nos modèles murins ont montré une tendance à la surexpression de nos gènes d’intérêts dans les tissus tumoraux des souris traités. En conclusion, ces résultats suggèrent l’implication de la voie de signalisation CXCL7/CXCR2 comme facteur prédictif de mauvais pronostic dans le cancer colorectal métastatique. Les chimiothérapies à base de 5 Fluoro-uracile augmentent l’expression de ces gènes dans les métastases hépatiques, fournissant une explication sur l’agressivité des tumeurs métastatiques en échappement thérapeutique. Un blocage sélectif de l’axe CXCR2/CXL7 pourrait fournir de nouvelles opportunités thérapeutiques. / Our aim was to analyze the potential role of chemokine receptors CXCR2 and CXCR4 signalling pathways in liver metastatic colorectal cancer (CRC) relapse. Expression levels of CXCR2, CXCR4, and their chemokine ligands were evaluated in liver metastases of colorectal cancer in order to study their correlation with overall and disease-free survival of patients having received, or not received, a neoadjuvant chemotherapy regimen.Quantitative RT-PCR and CXCR2 immunohistochemical staining were carried out using human CRC liver metastasis samples. Expression levels of CXCR2, CXCR4, and their ligands were statistically analyzed according to treatment with neoadjuvant chemotherapy and patients ' outcome. Murine models of subcutaneous and orthotopic intracaecal xenografts have been developed and used to study the expression of CXCR2, CXCR4 and CXCL7 in connection with the treatment of mice with chemotherapy.We showed that CXCR2 and CXCL7 overexpression are correlated to patient’s shorter overall and disease-free survival. By multivariate analysis, CXCR2 and CXCL7 expressions are independent factors of overall and disease-free survival. Neoadjuvant chemotherapy increases significantly the expression of CXCR2 and CXCL7 was overexpressed close to significance. Results of our mouse models have shown a trend over-expression of our interest genes in tumor tissues of the treated mice.In conclusion, we show the involvement of CXCL7/CXCR2 signalling pathways as a predictive factor of poor outcome in metastatic CRC. 5-Fluorouracil-based chemotherapy regimens increase the expression of these genes in liver metastasis, providing one explanation for aggressiveness of relapsed drug-resistant tumors. Selective blockage of CXCR2/CXCL7 signalling pathways could provide new potential therapeutic opportunities.

Heart transplantation (HTx) is now an established therapy for end-stage cardiac failure not responding to medical treatment. Recent decades have seen improved outcome following HTx due to more effective and targeted immunosuppressive therapy. However, acute and chronic rejection remains a major cause of morbidity and mortality. At the same time, immunosuppressive strategies are associated with significant side effects, including development of tumours. Hence, the induction of immunologic tolerance to alloantigen is considered the “holy grail” of transplant research. T regulatory cells (Tregs) are a subset of T cells that appear to suppresscytotoxic cell and initiate tolerance to foreign tissues. The Tregs suppresscytotoxic cells through specific cytokine pathways and cell-cell contact. In-vivo T reg migration has been a matter of debate in recent years. Treg trafficking is governed by chemokines, which are small secreted proteins, acting via their distinct trans-membrane serpentine receptors. Experimental work has demonstrated an involvement of distinct chemokine pathways in Tregs migration and localization following cardiac transplantation; however, there is paucity of data in humans. I investigated the effects of chemokines on Tregs in heart transplant recipients through a series of observational studies. My study demonstrated that acute rejection following heart transplantation is associated with a significant elevation of peripheral blood Th1 chemokine levels. I hereby further show that peripheral blood Treg counts in stable heart transplant recipients are not affected by immunosuppression but are significantly lower in patients taking statins. I have demonstrated via in-vitro chemotaxis assays a specific pattern of chemotactic response for Tregs and the effector T cells. Using double immunofluorescence staining and immunostaining, I show for the first time that Tregs may migrate to the allograft under the influence of CCL17.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 122-140).

Les récepteurs de chimiokines sont des régulateurs essentiels de la migration cellulaire dans le cadre de la surveillance immunitaire, et le développement. Les récepteurs CCR5 et CXCR4 sont de plus spécifiquement impliqués dans les métastases cancéreuses et l'infection par le VIH. Nous avons développé un système permettant de sur-exprimer ces deux RCPGs. Afin de s'affranchir des problèmes de toxicité inhérents à l'expression des protéines membranaires en bactérie notre approche de production consiste à adresser les protéines vers les corps d'inclusion d'E. coli grâce à une fusion protéique N-terminale permettant de hauts niveaux d'expression. Après purification en conditions dénaturantes, les protéines sont alors repliées en présence de surfactants originaux, les amphipoles. La validation de cette nouvelle approche pour les récepteurs des chimiokines représente un des objectifs principaux de ce travail. Afin de tester la fonctionnalité des protéines repliées, une série d'outils a été développée : des versions modifiées des chimiokines ont été produites (RANTES pour CCR5 et SDF 1a pour CXCR4). La fonctionnalité des chimiokines a été évaluée au niveau moléculaire et cellulaire. L'interaction entre le récepteur replié en amphipole et son ligand a été testé par résonance de plasmons de surface (SPR). Différents types de surfaces fonctionalisées avec le récepteur de chimiokine replié en amphipole ont été explorés au cours de ce travail. A la fin de ce projet, la production des chimiokines et de leur récepteur a été mise au point. L'accès à ces outils ouvre la voie à de futures études moléculaires telles que la compréhension de la dimérisation du récepteur ou la détermination de la stoechiométrie du complexe. / Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation and development. The GPCRs (G protein-coupled receptors) CCR5 and CXCR4 are specifically implicated in cancer metastasis and HIV-1 infection. An expression system to over-express these two GPCRs was developed. To overcome the toxicity problem of membrane protein expression in bacterial system, the production approach consists in targeting the proteins towards E. coli inclusion bodies thanks to a N-terminal fusion allowing a high yield expression. After purification under denaturing conditions, these GPCRs were then folded using original polymeric surfactants: the amphipols. The validation of this new approach for the chemokine receptor production is one of the goals of this work. In order to assess the functionality of the folded proteins, series of tools have been developed: engineered chemokine ligands (RANTES for CCR5 and SDF1a for CXCR4) were produced. The functionality of chemokines was evaluated at cellular and molecular levels. Interaction between the receptor folded in amphipols and its ligand was evaluated using Surface Plasmon Resonance (SPR) technique. Several types of surfaces, functionalized with the chemokine receptor/amphipol complex have been explored in this work. At the end of this project the productions of chemokines and their receptors has been set up. These established tools open the way to future studies, at the molecular level, in order to, for instance, investigate receptor dimerization and complex stoichiometry.

No description available.

Cellular senescence has been proposed to be equivalent to organismal aging and is one of the outcomes of the cell fate decision process in response to DNA damage that occurs in cells. When a cell encounters DNA damage, the cell cycle is immediately halted to evaluate which decision to take in response to genomic insult. The choices are between repairing the damage and continue division, or enter a non-replicative but viable state called senescence or to die if damage is severe (Figure 1). The signaling cascade, which detects this damage and regulates the cell fate decision, is collectively called as DNA damage response (DDR). However, the exact mechanism of how delineation for each decision happens is still not clear. Since DNA damage works as a mediator for cell fate decision, my work aimed to study senescence as a DNA damage response. In addition, the role of free radicals like ROS in cellular senescence is not very clear because though an increase in their concentrations is recorded in aged cells, it is not evident if the increase seen the cause or the effect of aging, primarily because they themselves capable of causing DNA damage. This conundrum have always led to confounding observations wrt role of free radicals in the cellular senescence process and if the senescence is caused through agents which rely on ROS to cause DNA damage, ROS becomes absolutely integral to the aging process. To understand this aspect formed the first line of investigation in my work along with identification of the sensor of DNA damage, which drives various cell fates. During organismal ageing there is an accumulation of senescent cells, which could be the major reason for functional decline of tissues and organs with age. However, to study changes associated with signaling molecules with respect to ageing, a cellular model system for senescence driven through DNA damage was needed, using which interplay between senescent / aged cells and cellular niche can be established. Studying the spatial and temporal alterations in signaling dynamics, within the cell as well as with the neighbouring niche during the senescence process in anticipated to provide us better understanding about the complex process of ageing. For this, the objectives were defined to establish and characterize the DNA damage induced senescence model using various parameters, and especially study the signaling dynamics of GPCR mediated signaling in senescence. The role of chemokine receptor, CXCR4 and its ligand, CXCL12 mediated signaling was chosen for the study. The following sections describe the findings that were obtained from the various objectives studied during the course of this study. Section 1. Development and characterization a model system to study cellular senescence as a DNA damage response. In this part of the study, I characterized genotoxic stress induced cellular senescence model using 5-Bromodeoxyrudine as the DNA damaging agent. BrdU, owing to its property of being a thymidine analogue, is incorporated in dividing cells, and this incorporation is recognized as DNA damage. This triggers ‘persistent’ DNA damage response signaling, including activation of ATM kinase, one of the primary DNA damage sensor. As anticipated, the DDR response detected was directly proportional to the dose of BrdU treatment and so was Reactive Oxygen Species (ROS) levels, a known senescence mediator. Using this model system of direct DNA damage mediated DDR activation and induction of cellular senescence, the growth-arrested cells were extensively characterized for presence and quantum of most of the senescence associated markers known in literature. BrdU treated cells, which became senescent showed presence of DNA damage, morphological changes like flat, enlarged, granule rich appearance, expression of senescence associated molecular markers like p21, IL8, showed senescence associated beta galactosidase activity, refractiveness to growth factor for division, increased ROS levels, Golgi dispersion, etc. The secretome of the treated cells also showed increased secretion of inflammatory cytokines which are attributed to a senescence phenotype, called as Senescence Associated Secretory Phenotype (SASP), which triggered proliferative and migratory effect on cancer cells. Overall, in this part of the study, it was established that BrdU can cause DNA damage and induce senescence as one of the cell fate in response to the intermediate dose of damage. The senescent cells generated in the model system was established to be akin to senescence observed by replicative exhaustion of normal cells, thereby making our model applicable to the physiological studies as well. Section 2. Insights into the role of ATM-ROS axis during senescence initiation and maintenance using DDR mediated cellular senescence model. While the BrdU model system for generating senescent cells was being developed and characterized, it was observed that there is an increase in ATM activation as well as ROS production concomitant to the a dose of BrdU. At the same time it was also observed that senescent cells showed persistent DDR signaling and high levels of ROS. Using this premise, in the second objective of my study I aimed to identify if ATM and ROS are critical during initiation of senescence, when the cells are insulted with the DNA damaging agent or during the maintenance of senescent state of the cells. By quenching ROS during the initiation state, I recorded that ROS is not critical for inducing senescence and perhaps the increase in ROS levels in senescent cells is due to their higher metabolic activity. By inhibiting ATM activation during DNA damage, it was observed that BrdU induces senescence through direct DNA damage, and active ATM and DDR signaling is absolutely critical for the senescence initiation. It was also established that ATM is not just a DNA damage sensor but also a redox regulator in the senescence model system. Prevention of ATM activation in presence of DNA damage blocked senescence initiation and also triggered increased ROS levels in the cells affecting their long term viability, suggesting ATM regulates ROS levels as well in addition to sensing DNA damage. In order to study the role of ATM-ROS axis in the maintenance of senescence state, already senescent cells were subjected to ROS quenching and/ or ATM inhibition and it was identified that both these signaling molecules are essential for maintaining the viability of senescent cells. The findings from these study thereby show that senescence can be divided into two temporally distinct stages, initiation or early senescence stage and second, maintenance stage of senescence. Overall, I was able to characterize the presence of temporally linked ROS – dependent and ROS – independent events in cellular senescence, which are independently mediated by ATM kinase (Figure 1). Dose of Genotoxic Stress damage DDR Senescence initiation Repair Cell cycle ATM arrest kinase Death Growth arrest Senescence maintenance Senescence Cell ROS viability Elevated metabolism Figure 1. Signaling cascades regulating senescence onset and maintenance mediated through DDR. Cells enter senescence state in response to DNA damage, depending on the dose of insult, through an ATM dependent and ROS independent pathway. Unlike this ATM-ROS axis is critical for the maintenance of senescent state of damaged but viable cells. Section 3. Understanding the role of CXCR4 – CXCL12 mediated signaling in senescence. Age dependent changes in cellular signaling are less explored and I was specifically interested in understanding how presence of senescent cells affects its microenvironment or vice versa i.e. how microenvironment affects senescent cells. In this premise the third objective of this study was defined towards identifying role of a GPCR, CXCR4 mediated signaling in cellular senescence and associated inflammation. CXCR4 is a ubiquitously expressed GPCR and it’s only known ligand is CXCL12/ SDF1 (stromal derived factor ), which is a homeostatic chemokine (i.e. its levels does not change under most physiological conditions). During characterization of DNA damage induced senescence model system, it was observed that this receptor expression is induced during DNA damage ells, which was also found to be so from data available from other gene expression studies as well. During the course of my work, I identified that senescent cells show CXCR4 up regulation in response to DNA damage, mediated through activation of ATM kinase - HIF1 axis and plays a critical role in enhancing the senescence associated inflammatory response in presence of its ligand, CXCL12. This CXCL12 dependent enhanced inflammatory response in damaged cells was determined to be sensitive to the pertussis toxin treatment and hence dependent on G protein activation. Further downstream analysis revealed the pro-inflammatory effect of the CXCR4 receptor activation was due to cAMP level suppression post activation by the Gi subunit. Given that cAMP levels are antagonistic to inflammatory phenotype, using a library of pharmacological compound library, I also discovered that cAMP specific PDE, phophodiesterase 4A, is also involved in regulating inflammatory response during the initiation stage of cellular senescence. The screen also confirmed the involvement of previously identified molecular components such as p38 MAPK and leukotrienes in the senescence associated inflammatory phenotype. The examination of the role of the CXCR4- CXCL12 axis in the deeply senescent cells surprisingly revealed that deeply senescent cells are refractory to CXCL12 stimulation in terms of inflammatory response, which was experimentally determined to be associated with impaired calcium release. Overall, the findings from this part of the study revealed a novel signaling cascade where CXCR4 up regulation is a part of the DDR response in cells, which utilizes the Local Excitation Global Inhibition (LEGI) mechanism to enhance the sensitivity of the damaged cells to its ligand CXCL12. This enhanced sensitivity mediates the CXCL12 dependent inflammatory response, which aids in attracting immune cells for clearance of these damaged cells. Once the cells have entered the senescent state, the axis is physiologically down modulated and the senescent cells showed refractiveness to CXCL12 stimulation, probably to prevent persistent acute inflammation, if the senescent cells are not cleared (Figure 2). Figure 2. CXCL12-CXCR4 axis in cellular senescence. During senescence initiation stage, when cells encounter DNA damage (Step 1), there is induction of CXCR4 receptor (Step 2), which enhances of CXCL12 mediated signaling for increased inflammatory response (Step 3). In the maintenance stage, where the cells are not cleared (Step 4), the axis is suppressed (Step 4), thereby bringing the levels of inflammatory secretome down, and thereby preventing damage to the cells (Step 5).