Le récepteur de chimiokine CXCR7 a été récemment identifié comme liant la chimiokine SDF-1, anciennement considérée comme ligand exclusif du récepteur CXCR4. Ces deux récepteurs sont exprimés majoritairement dans les mêmes types cellulaires et, ainsi, la découverte de CXCR7 incite à réévaluer les effets respectifs de SDF-1 sur CXCR4. Étant donné son rôle dans le cancer, CXCR4 est une cible de choix pour le développement de molécules thérapeutiques. Également, CXCR7 semble être impliqué dans la croissance tumorale. AMD3100, un antagoniste «sélectif» pour CXCR4, est maintenant commercialisé. Cet antagoniste a été identifié comme liant lui aussi CXCR7. De plus, sur CXCR7, l’AMD3100 agit comme agoniste puisqu’il induit le recrutement de la β-arrestine, à l’opposé de son effet sur. En revanche, AMD3100 n’induit pas le recrutement de la β-arrestine à CXCR4. Basé sur ces résultats, il est nécessaire de revoir la sélectivité d’autres antagonistes synthétiques de CXCR4. À l’aide de la technique de BRET (Résonance d’un transfert d’énergie par bioluminescence), nos résultats montrent que le Tc14012, un autre antagoniste synthétique de CXCR4, et structurellement distinct de l’AMD3100, interagit avec CXCR7. Contrairement à CXCR4, les deux antagonistes de CXCR4 agissent comme agonistes sur CXCR7 en induisant le recrutement de la β-arrestine. Nos résultats suggèrent que l’organisation spatiale du corps du récepteur serait responsable de cet effet opposé. En conclusion, AMD3100 et Tc14012 ne sont pas sélectifs pour CXCR4, puisqu’ils interagissent avec CXCR7. Lors du développement de nouvelles molécules synthétiques ciblant CXCR4, il serait alors nécessaire d’en évaluer leur sélectivité, et leurs effets en les testant aussi sur CXCR7. / ASBTRACT SDF-1 was at first thought to exclusively bind CXCR4, but it was subsequently found to also bind to the chemokine receptor CXCR7. CXCR4 is a promising target for drug development due to its role in cancer. AMD3100 is newly commercialised synthetic antagonist of CXCR4. This drug leads to massive release of hematopoietic stem cell into the peripheral blood. It was found that AMD3100 also binds to CXCR7 and acts as an agonist of β-arrestin recruitment to CXCR7. An antagonist of CXCR4 acts as an agonist on CXCR7. Prompted by this observation, we tested whether this might hold true for other CXCR4 antagonist. Tc14012, a peptidomimetic of T140, has been extensively described as a potent CXCR4 antagonist. We find that TC14012 also interacts on CXCR7. Like AMD3100, TC14012 alone induces β-arrestin recruitment to CXCR7. Thus, two structurally unrelated CXCR4 antagonists, AMD3100 and TC14012, are agonists of the CXCR7-arrestin pathway. This suggests distinct activation mechanisms of the arrestin pathway by CXCR4 and CXCR7. The results we obtained using a BRET (Bioluminescence Resonance Energy Transfer)-based arrestin recruitment assay, suggest that the CXCR7 receptor core is responsible for the recruitment of beta-arrestin in response to AMD3100 and TC14012. The finding that both AMD3100 and TC14012 do not only bind CXCR4, but also CXCR7, with opposite effects on arrestin recruitment, is important for the use of the compounds as tools to dissect SDF-1-mediated effects. This may be a general feature of synthetic ligands of the two receptors, with potential consequences for drug development. Key words: Chemokine receptor, CXCR4 and CXCR7, BRET, β-arrestin recruitement, TC14012, AMD3100 and SDF-1.

No description available.

RÉSUMÉ Le récepteur de chimiokine CXCR7 a été récemment identifié comme liant la chimiokine SDF-1, anciennement considérée comme ligand exclusif du récepteur CXCR4. Ces deux récepteurs sont exprimés majoritairement dans les mêmes types cellulaires et, ainsi, la découverte de CXCR7 incite à réévaluer les effets respectifs de SDF-1 sur CXCR4. Étant donné son rôle dans le cancer, CXCR4 est une cible de choix pour le développement de molécules thérapeutiques. Également, CXCR7 semble être impliqué dans la croissance tumorale. AMD3100, un antagoniste «sélectif» pour CXCR4, est maintenant commercialisé. Cet antagoniste a été identifié comme liant lui aussi CXCR7. De plus, sur CXCR7, l’AMD3100 agit comme agoniste puisqu’il induit le recrutement de la β-arrestine, à l’opposé de son effet sur. En revanche, AMD3100 n’induit pas le recrutement de la β-arrestine à CXCR4. Basé sur ces résultats, il est nécessaire de revoir la sélectivité d’autres antagonistes synthétiques de CXCR4. À l’aide de la technique de BRET (Résonance d’un transfert d’énergie par bioluminescence), nos résultats montrent que le Tc14012, un autre antagoniste synthétique de CXCR4, et structurellement distinct de l’AMD3100, interagit avec CXCR7. Contrairement à CXCR4, les deux antagonistes de CXCR4 agissent comme agonistes sur CXCR7 en induisant le recrutement de la β-arrestine. Nos résultats suggèrent que l’organisation spatiale du corps du récepteur serait responsable de cet effet opposé. En conclusion, AMD3100 et Tc14012 ne sont pas sélectifs pour CXCR4, puisqu’ils interagissent avec CXCR7. Lors du développement de nouvelles molécules synthétiques ciblant CXCR4, il serait alors nécessaire d’en évaluer leur sélectivité, et leurs effets en les testant aussi sur CXCR7. Mot-clés : Récepteur de chimiokine CXCR4 et CXCR7, BRET, recrutement de la β-arrestine, AMD3100, Tc14012 et SDF-1. / ASBTRACT SDF-1 was at first thought to exclusively bind CXCR4, but it was subsequently found to also bind to the chemokine receptor CXCR7. CXCR4 is a promising target for drug development due to its role in cancer. AMD3100 is newly commercialised synthetic antagonist of CXCR4. This drug leads to massive release of hematopoietic stem cell into the peripheral blood. It was found that AMD3100 also binds to CXCR7 and acts as an agonist of β-arrestin recruitment to CXCR7. An antagonist of CXCR4 acts as an agonist on CXCR7. Prompted by this observation, we tested whether this might hold true for other CXCR4 antagonist. Tc14012, a peptidomimetic of T140, has been extensively described as a potent CXCR4 antagonist. We find that TC14012 also interacts on CXCR7. Like AMD3100, TC14012 alone induces β-arrestin recruitment to CXCR7. Thus, two structurally unrelated CXCR4 antagonists, AMD3100 and TC14012, are agonists of the CXCR7-arrestin pathway. This suggests distinct activation mechanisms of the arrestin pathway by CXCR4 and CXCR7. The results we obtained using a BRET (Bioluminescence Resonance Energy Transfer)-based arrestin recruitment assay, suggest that the CXCR7 receptor core is responsible for the recruitment of beta-arrestin in response to AMD3100 and TC14012. The finding that both AMD3100 and TC14012 do not only bind CXCR4, but also CXCR7, with opposite effects on arrestin recruitment, is important for the use of the compounds as tools to dissect SDF-1-mediated effects. This may be a general feature of synthetic ligands of the two receptors, with potential consequences for drug development. Key words: Chemokine receptor, CXCR4 and CXCR7, BRET, β-arrestin recruitement, TC14012, AMD3100 and SDF-1.

No description available.

Microscopic colitis (MC), comprising collagenous colitis (CC) and lymphocytic colitis (LC) is a common cause of chronic watery diarrhea. The diagnosis relies on typical histopathological changes observed upon microscopic examination. The studies in this thesis investigated innate and adaptive immune responses in the colonic mucosa of MC patients, also comparing patients with active disease (CC and LC) and histopathologically in remission (CC/LC-HR). We first analyzed expression of interleukin-1/Toll-like receptor (IL-1/TLR) signaling regulators in MC patients (Paper I). Our results showed enhanced IRAK-M, microRNA-146a, -155 and -21 expressions, whereas IL-37 gene expression was reduced in CC and LC patients as compared to non-inflamed controls. These results suggest different pathophysiological mechanisms in MC patients. The mixed inflammatory cell infiltrations seen in the lamina propria of MC patients might be a result of dysregulated expression of chemotactic mediators. In Paper II, we showed that MC patients display mainly an increased expression of chemokines and chemokine receptors in active disease as compared to noninflamed controls. In Paper III, we examined if the decreased IL-37 expression seen in Paper I could mediate the upregulation of chemokines seen in Paper II. We showed that a relatively small reduction in the ability of epithelial cells to produce IL-37 results in mainly increased chemokine expressions in a pattern similar to the findings in Paper II. In order to understand the nature of infiltrating T cells commonly observed in MC patients, we analyzed the T cell receptor (TCR) β chains in colonic biopsies of MC patients (Paper IV). Our results showed significant differences in TCRβ repertoire, which suggests selectively expanded T cell clones in active MC and histopathologically in remission patients. Altogether, these results i) increase the knowledge of MC pathogenesis by showing changes in TLR signaling regulators, enhanced chemokine and their receptor expressions involved in a mixed immune cell infiltrations and selectively expanded T cell clones in CC and LC patients, as well as in histopathological remission ii) might potentially increase the possibility of more target-specific therapies based on IL-37 induction, chemokines or chemokine receptor inhibitions, or hindering T cell infiltration according to TCR clonality.

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).

Exogenous and endogenous cannabinoids have been reported to modulate functional activities of macrophages. It is recognized that macrophages express primarily the CB2 cannabinoid receptor, but recent studies indicate that its expression is differential in relation to activation state with maximal levels occurring when cells are in "responsive" and "primed" states. The functional activities of macrophages when in these states of activation are the most susceptible to the action of cannabinoids, at least in terms of a functional linkage to the CB2. To assess the effect of cannabinoid treatment on macrophage chemotaxis and test the hypothesis that cannabinoids inhibit the chemotactic response of macrophages and microglia to endogenous and exogenous, pathogen-derived stimuli, primary murine peritoneal macrophages and neonatal rat microglia were used. Chemotaxis assays and scanning electron microscopy studies demonstrated that cannabinoids inhibit chemotaxis, a signature activity attributed to "responsive" macrophage-like cells, to the endogenous chemokine RANTES (Regulated upon Activation Normal T-cell Expressed and Secreted) and to Acanthamoeba conditioned medium containing secreted proteases. The partial agonist delta-9-tetrahydrocannabinol (THC), administered in vitro, inhibited the chemotactic response of peritoneal macrophages to the chemokine RANTES and to Acanthamoeba conditioned medium. In vivo treatment with THC also resulted in inhibition of the in vitro chemotactic response of murine peritoneal macrophages to RANTES and amoebic conditioned medium. Pharmacological studies employing cannabinoid receptor agonists and antagonists demonstrated the involvement of CB2 in cannabinoid-mediated inhibition of peritoneal macrophage chemotaxis to RANTES and Acanthamoeba conditioned medium, implying that signaling through cannabinoid receptors may desensitize chemokine receptors. Treatment with cannabinoids had no apparent effect on chemokine receptor mRNA levels, but did enhance CCR5 protein phosphorylation. Macrophage migration to Acanthamoeba conditioned medium may involve activation and signaling through protease activated receptors (PARs), as pathogen-derived proteases have been shown to activate PARs and initiate cellular migration; however, further studies are required to demonstrate PAR activation by amoebic conditioned medium and to assess the effects of cannabinoids on PAR signaling. Acanthamoeba are opportunistic pathogens that cause Granulomatis amoebic encephalitis, an infection of the CNS that is often fatal. THC treatment has been shown to increase mortality to Acanthamoeba infections and is characterized by an absence of granuloma formation. We hypothesize that inhibitory effect of THC on macrophage migration may be a key factor in cannabinoid-mediated immunosuppression. To assess the effect of cannabinoids on microglial migration to Acanthamoeba conditioned medium, chemotaxis assays were performed using primary rat microglia treated with cannabinoids. These studies demonstrated that cannabinoids inhibit microglial chemotaxis to amoebic conditioned medium. Furthermore, the studies demonstrate that cannabinoids, acting through cannabinoid receptors, may cross-talk with a diverse array G-protein coupled receptors so as to modulate responsiveness of macrophage and macrophage-like cells.

Head and neck squamous cell carcinoma is the 6th most common malignancyworldwide. Recently, a link between cancer and inflammation has been found. Mediatingthis relationship are the chemotactic cytokines known as chemokines. CXCL8 (Interleukin-8), a CXC ELR+ Chemokine mainly responsible for neutrophil chemoattraction, has beenimplicated in increased tumor proliferation, migration and angiogenesis. The current studytests the effects of CXCL8 on the tumor proliferation and metastasis. By genetically modifying cells to knockdown or overexpress the CXCL8 gene we tested its biological rolein head and neck cancer progression. Overexpression of CXCL8 in HN4 tumor cells withlow endogenous CXCL8 levels was found to increase tumor growth, as judged by cellcounting and MTT assays. Conversely, RNAi-mediated knockdown of CXCL8 expressionin HN12 cells, which express high levels of this chemokine, resulted in a decrease inproliferation. Similarly, overexpression of CXCL8 enhanced migration of HN4 cells invitro, while knockdown inhibited HN12 cell migration and invasion through a basementmembrane substitute. Taken together, these findings support the hypothesis that CXCL8affects multiple processes involved in head and neck cancer tumor progression. The datasuggest that CXCL8 is a potential therapeutic target for head and neck, and other, cancers.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is associated with formation of autoantibodies, activation of inflammatory cascade and up-regulation of several cytokines. These processes lead to persistent synovial inflammation, joint damage and systemic manifestations. The aim of this diploma thesis is to characterize the role of a novel cytokine interleukin-20 (IL-20) in the pathogenesis of RA and to investigate its involvement in different stages of the disease as a potential surrogate biomarker. In this work, several methods including Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry and Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) have been employed. We demonstrated increased expression of IL-20 in the synovial tissue of RA compared with control osteoarthritis (OA) patients. Along with the up-regulation at sites of inflammation, concentrations of IL-20 were higher in the synovial fluid compared with circulating levels of IL-20. Furthermore, serum and synovial fluid IL-20 levels significantly correlated with RA disease activity. Synthesis of IL-20 was significantly increased in peripheral blood mononuclear cells (PBMCs) and synovial fibroblasts upon stimulation with some TLR ligands and pro-inflammatory cytokines. Although not regulating PBMCs functions in...

Charcot-Marie-Tooth 1B (CMT1B) is a progressive inherited demyelinating disease of human peripheral nervous system leading to sensory and/or motor function disability and is caused by mutations in the P0 gene. Mice heterozygously deficient for P0 (P0+/-) are an adequate model of this human disorder showing myelin degeneration, formation of onion bulbs, remyelination and a reduced motor conduction velocity of around 30m/s similar to patients. Previously, it had been shown that T-lymphocytes and macrophages play a crucial role during pathogenesis in peripheral nerves of P0+/- mice. Both, T-lymphocytes and macrophages increase in number in the endoneurium and deletion of T-lymphocytes or deletion of a macrophage-directed cytokine ameliorates the disease. In this study the monocyte chemoattractant protein-1 (MCP-1) was identified as an early regulated cytokine before onset of disease is visible at the age of six months. MCP-1 mRNA and protein expression could be detected in femoral quadriceps and sciatic nerves of P0+/- mice already at the age of one month but not in cutaneous saphenous nerves which are never affected by the disease. MCP-1 was shown to be expressed by Schwann cells and to mediate the immigration of immune cells into peripheral nerves. Deletion of MCP-1 in P0+/- mice accomplished by crossbreeding P0 and MCP-1 deficient mice revealed a substantial reduction of immune cells in peripheral nerves of P0+/-/MCP-1+/- and P0+/-/MCP-1-/- mice at the age of six months. In twelve months old mice reduction of immune cells in peripheral nerves is accompanied by amelioration of demyelinating disease in P0+/-/MCP-1+/- and aggravation of demyelinating disease in lumbar ventral roots of P0+/ /MCP-1-/- mice in comparison to P0+/ /MCP 1+/+ mice. Furthermore, activation of the MEK1/2-ERK1/2 signalling cascade could be demonstrated to take place in Schwann cells of affected peripheral nerves of P0+/- mice overlapping temporarily and spatially with MCP-1 expression. An animal experiment using a MEK1/2-inhibitor in vivo, CI-1040, revealed that upon reduction of ERK1/2 phosphorylation MCP-1 mRNA expression is diminished suggesting that the activation of the MEK1/2-ERK1/2 signalling cascade is necessary for MCP-1 expression. Additionally, peripheral nerves of P0+/- mice showing reduced ERK1/2 phosphorylation and MCP-1 mRNA expression also show reduced numbers of macrophages in the endoneurium. This study shows a molecular link between a Schwann cell based mutation and immune cell function. Inhibition of the identified signalling cascade might be a putative target for therapeutic approaches. / Die humane Erkrankung Charcot-Marie-Tooth 1B (CMT1B) ist eine erbliche, chronisch fortschreitende Erkrankung des peripheren Nervensystems die durch Mutation des P0-Gens verursacht wird und zu motorischen und/oder sensorischen Defiziten führt. Sehr ähnlich der humanen Erkrankung weist das Mausmodell, eine für das Myelinprotein P0 heterozygot-defiziente Maus (P0+/-), Degeneration peripheren Myelins, aufeinanderfolgende Zyklen von De- und Remyelinisierung als auch reduzierte Nervenleitgeschwindigkeiten auf. Wissenschaftliche Untersuchungen am Mausmodell ergaben eine Beteiligung von T-Lymphozyten und Makrophagen an der Pathogenese. In dieser Studie wurde das Chemokin „Monocyte Chemoattractant Protein-1“ (MCP-1) als pathogen-relevant in P0+/- Mäusen identifiziert. MCP-1 mRNA und Protein wurden sowohl im Alter von sechs und zwölf Monaten nachgewiesen, Stadien, in denen morphologische Veränderungen peripherer Nerven von P0+/- Mäusen zu erkennen sind, aber auch im Alter von einen und drei Monaten, ein Alter bei dem pathologischen Veränderungen nicht zu finden sind. Mit Hilfe von MCP-1 defizienten Mäusen (MCP-1-/-) und Verpaarung mit P0-defizienten Mäusen konnten weiterführende Untersuchungen zur Rolle von MCP-1 im peripheren Nerv der Maus durchgeführt werden. So zeigte es sich mittels Transplantation von GFP-positivem Knochenmark, dass MCP 1 die Infiltration von Makrophagen aus dem Blut in periphere Nerven vermittelt. Weiterhin konnte gezeigt werden, dass periphere Nerven von sechs Monate alten P0+/-/MCP-1+/- und P0+/-/MCP-1-/- Mäusen trotz signifikant niedrigerer Anzahl von Immunzellen keine Milderung der Demyelinisierung zeigen. Hingegen weisen periphere Nerven von zwölf Monate alten P0+/ /MCP-1+/- Mäusen sowohl weniger Makrophagen und T-Lymphozyten als auch wesentlich weniger pathologische Veränderungen auf. Periphere Nerven von P0+/-/MCP-1-/- Tieren dagegen zeigen nur eine nicht signifikante Reduktion von Immunzellen und sogar eine Verschlechterung des Phänotyps im Vergleich zu ventralen Spinalwurzeln von P0+/-/MCP-1+/+ Mäusen. Weiterführende Untersuchungen ergaben, dass eine Aktivierung der MEK1/2-ERK1/2 Signalkaskade sowohl in peripheren Nerven von drei und sechs Monate alten P0+/- Mäusen zu finden ist, allerdings, ähnlich der Expression von MCP-1, nur in peripheren Nerven, die von der Demyelinisierung betroffen sein können. Unter Verwendung eines Inhibitors der Kinasen MEK1 und 2 konnte in vivo gezeigt werden, dass Phosphorylierung von ERK1/2 für die erhöhte MCP-1 Expression in peripheren Nerven von P0+/- Mäusen notwendig ist. Darüber hinaus wurde durch Verminderung der ERK1/2-Phosphorylierung eine Reduktion von Makrophagen im Endoneurium von P0+/- Tieren erzielt.