Chronic kidney disease (CKD) is associated with a complex state of immune dysfunction characterised by immune depression, which predisposes CKD patients to infections, and by immune activation resulting in inflammation, which is associated with cardiovascular disease among these patients. It has been suggested that regular moderate exercise may enhance immune function and exert anti-inflammatory effects. However, such effects are still unclear in CKD. Therefore, we investigated the effects of acute and regular (1-month and 6-months) moderate intensity aerobic exercise (walking) on measures of immunity and inflammation in pre-dialysis CKD patients. A single bout of walking exercise induced an overall immune and inflammatory response that was comparable to that observed in healthy individuals, with no indication of harmful effects to patients underlying state of immune dysfunction. Acute exercise induced a normal pattern of mobilisation of immune cells. Concerning immune cell function, acute exercise had no effect on T lymphocyte and monocyte activation, while it actually improved neutrophil responsiveness to a bacterial challenge in the recovery period. In addition, acute exercise induced a systemic anti-inflammatory environment, evidenced by the marked elevation in plasma IL-10 levels after exercise, which was most likely mediated by the observed increase in plasma IL-6 levels. Regular walking exercise exerted anti-inflammatory effects, with no apparent detrimental effects to patients immune and inflammatory status. Regular exercise led to improvements in the systemic inflammatory status (ratio of pro-inflammatory IL-6 to anti-inflammatory IL-10 cytokine levels) that were accompanied, and most likely mediated, by the observed down-regulation of T lymphocyte (only evident at 6-months) and monocyte activation. In addition, a reduction in IL-6 production in PBMC and whole blood cultures was also observed (only assessed at 1-month). Regular exercise had no effect on circulating immune cell numbers and neutrophil degranulation responses. These findings provide compelling evidence that walking exercise is safe from an immune and inflammatory perspective and has the potential to be an effective anti- inflammatory therapy in pre-dialysis CKD patients.

Thromboxane A2 receptor (TP) has been shown to play important roles in multiple aspects of cancer development including regulation of tumor growth, survival and metastasis. Molecular mechanisms of TP mediated cancer cell invasion remain to be identified. TP agonist, I-BOP, significantly elevated several matrix metalloproteinases (MMPs) including MMP-1, MMP-3, MMP-9 and MMP-10 in A549 human lung adenocarcinoma cells overexpressing TPα (A549-TPα) or TPβ (A549-TPβ). Signaling pathways of I-BOP-induced MMP-1 expression were examined in further detail as a model system for MMPs induction. Signaling molecules involved in I-BOP-induced MMP-1 expression were identified by using specific inhibitors including small interfering (si)-RNAs of signaling molecules and promoter reporter assay. The results indicate that I-BOP-induced MMP-1 expression is mediated by protein kinase C (PKC), extracellular signal-regulated kinase (ERK)-activator protein-1(AP-1) and ERK-CCAAT/enhancer-binding protein β (C/EBPβ) pathways. I-BOP-induced cellular invasiveness of A549-TPα cells was blocked by, GM6001, a general inhibitor of MMPs. Knockdown of MMP-1 and MMP-9 by their respective siRNA partially reduced I-BOP-stimulated A549-TPα cells invasion suggesting that other MMPs induced by I-BOP were also involved. Furthermore, secreted MMP-1 in conditioned media from I-BOP-treated A549-TPα cells (CM-I-BOP) autocrinely induced monocyte chemoattractant protein-1 (MCP-1) expression. The induction of MCP-1 by MMP-1 in A549 cells was via activation of protease-activated receptor 2 (PAR2) instead of commonly assumed PAR1. This conclusion was reached from the following findings: (1) expression of MCP-1 induced by trypsin, a PAR2 agonist, was inhibited by a PAR2 antagonist. (2) expression of MCP-1 induced by MMP-1 and by CM-I-BOP was blocked by a PAR2 antagonist but not by other PAR antagonists; (3) expression of MCP-1 induced by MMP-1 and by CM-I-BOP was attenuated significantly by pretreatment of cells with PAR2-siRNA. Finally, MCP-1 also can be induced by direct activation of TP in a SP1 involved mechanism. CM-I-BOP enhanced MCP-1-dependent migration of RAW 264.7 macrophages. Co-culture of A549 cells with RAW 264.7 macrophages induced expression of MMPs, VEGF and MCP-1 genes, and increased the invasive potential in A549 cells. My studies provide molecular mechanisms by which TP-mediated cancer cell invasion and suggest that TP is a potential anti-cancer drug target.

<p>Myocardial infarction (MI) is often caused by a disruption of an atherosclerotic plaque with activation of coagulation, platelets and inflammation. The aims were; to investigate whether the oral direct thrombin inhibitor, ximelagatran affected markers for coagulation, platelet and inflammation in a patient cohort with recent MI and if the coagulation markers could identify patients with increased risk of new ischemic events; to evaluate some of the mechanisms involved in formation of platelet-monocyte aggregates (PMAs). </p><p>In a biomarker substudy patients with recent MI were randomized to 24-60 mg of ximelagatran or placebo for six months. There was a persistent dose-independent reduction of coagulation markers (F1+2, D-dimer) by ximelagatran treatment. 60 % reduced their D-dimer levels after one week and that group had less ischemic events during treatment. There was an early increase of the platelet activation marker and ximelagatran in higher doses attenuated these increased levels. Both in vivo and in vitro the direct thrombin inhibitor diminished procoagulant activity and tissue factor (TF) presenting microparticles. In contrast, the inflammatory markers increased after six months of ximelagatran treatment. The PMA-levels were elevated for long-term after MI. In vitro thrombin inhibition diminished formation of PMAs. Formation of PMAs in stimulated whole blood was P-selectin dependent and induced TF expression through phosphorylation of the Src-family member Lyn in monocytes.</p><p>Addition of an oral direct thrombin inhibitor reduces coagulation and platelet activation markers for long-term after a MI together with reduced procoagulant activity which may contribute to the clinical benefit of the drug. Early reduction of D-dimer levels seems to be suitable to identify patients with reduced risk of new ischemic events independent of antithrombotic treatment. Circulating PMAs persist after a MI connecting coagulation to inflammation. Within these aggregates P-selectin induces TF, the main initiator of coagulation, partly through phosphorylation of Lyn.</p>

Monocytic human leukocyte antigen-D related (mHLA-DR) expression determined by flow cytometry has been suggested as a biomarker of sepsisinduced immunosuppression. In order to facilitate use of HLA-DR in clinical practice, a quantitative real-time PCR technique measuring HLA-DR at the transcription level was developed and evalutated. Levels of HLA-DR mRNA correlated to mHLADR expression and were robustly measured, with high reproducibility, during the course of infection. Dynamics of mHLA-DR expression was studied during the first weeks of bloodstream infection (BSI) and was found to be dependent on the bacterial etiology of BSI. Moreover, mHLA-DR was shown to be inversely related to markers of inflammation. In patients with unfavourable outcome, sustained high C-reactive protein level and high neutrophil count were demonstrated along with low mHLA-DR expression and low lymphocyte count. This supports the theory of sustained inflammation in sepsis-induced immunosuppression. The association between mHLA-DR and bacterial etiology may be linked to the clinical trajectory via differences in ability to cause intractable infection. Staphylococcus aureus was the dominating etiology among cases with unfavourable outcome. With focus on patients with S. aureus BSI, those with complicated S. aureus BSI were found to have lower HLA-DR mRNA expression during the first week than those with uncomplicated S. aureus BSI. If these results can be confirmed in a larger cohort, HLA-DR measurement could possibly become an additional tool for early identification of patients who require further investigation to clear infectious foci and achieve source control. In conclusion, PCR-based measurement of HLA-DR is a promising method for measurements of the immune state in BSI, but needs further evaluation in the intensive care unit setting to define the predictive and prognostic value for deleterious immunosuppression. The etiology of infection should be taken into consideration in future studies of translational immunology in sepsis.

Les chimiokines (CKs) jouent un rôle important dans l’orchestration de la réponse immunitaireen contrôlant notamment la mobilisation des cellules immunitaires. Les cellules myéloïdes et, enparticulier, les monocytes sont impliquées dans le processus inflammatoire et notamment dansle développement des cancers. En effet, les cellules dérivées des monocytes ou macrophagesassociés aux tumeurs sont fortement représentés dans le micro environnement tumoral et sontsouvent associés à un mauvais pronostic. La caractérisation des mécanismes aboutissant aurecrutement des monocytes dans la tumeur représentent donc un enjeu majeur pourl’optimisation des protocoles thérapeutiques anti-cancéreux. Chez la souris, deux populationsmonocytaires sont caractérisées sur la base de l’expression différentielle des récepteurs auxchimiokines CCR2 et CX3CR1 et interviennent dans leur recrutement et leur différenciation enmacrophages dans les tissus, les monocytes inflammatoires (CCR2+, CX3CR1low) et les monocytesdits résidents (CCR2-, CX3CR1high). L’objectif principal de mon projet de recherche a été de mieuxcomprendre les mécanismes contrôlant la mobilisation des monocytes suite à un traitementchimiothérapeutique. J’ai entrepris d’étudier le rôle des récepteurs aux chimiokines, enparticulier CX3CR1, dans la reconstitution monocytaire après traitement chimiothérapeutiquepar le cyclophosphamide (CP), un agent alkylant reconnu pour son activité myélosuppressive. LeCP provoque un renouvellement des monocytes et une forte infiltration de la tumeur par les LTspécifiques de la tumeur (issus d’un transfert adoptif) associés à la réactivation de la réponseimmune anti-tumorale. Cependant, les LTs spécifiques de l’antigène de la tumeur se localisentpréférentiellement dans des zones riches en cellules dendritiques associées à la tumeur (TuDCs)et sont piégés par ces dernières. Ces interactions diminuent potentiellement le nombre decontacts entre les LT et les cellules tumorales suggérant un rôle pro tumoral des TuDCs. Letraitement au CP provoque une déplétion des cellules myéloïdes suivie d’une reconstitutionmassive des réservoirs de monocytes (moelle osseuse et rate). Au cours de la reconstitutionmonocytaire, l’expression de CX3CR1 diminue et est corrélée à une diminution de l’adhérence exvivo des cellules médullaires. Nous avons mis en évidence une mobilisation accrue desmonocytes inflammatoires au sein des souris CX3CR1- /- comparée aux souris WT et CCR2-/-.L’imagerie in vivo de la moelle osseuse au sein de souris CX3CR1-/- ou à l’aide d’un antagonistede CX3CR1 nous a permis de montrer un rôle spécifique de CX3CR1 dans le « crawling » sur lescellules endothéliales et le confinement des cellules monocytaires au niveau des sinus et duparenchyme médullaire. Nous suggérons qu’au cours de la mobilisation cellulaire induite par leCP le récepteur CX3CR1 contrôle la rétention médullaire des monocytes. Nous pensons que lamodulation du taux de mobilisation cellulaire au cours de la reconstitution induite par CP et/oule ciblage de CX3CR1 pourrait, par augmentation du pool de cellules myéloïdes leucocytairesd’un hôte, contribuer à l’amélioration des réponses cellulaires à la suite d’une lésion tissulaireou d’un dysfonctionnement des défenses immunitaires. De plus, le ciblage de CX3CR1 pourraittrouver des applications dans le domaine de la greffe de HSCs. / Chemokines orchestrate immune response especially by leucocytes mobilization. Myeloidcells, notably monocytes, are involved in inflammation and cancer development. Indeedmonocyte-derived cells and macrophages are strongly represented in tumourmicroenvironment and are associated with a bad prognosis. Characterization of mechanismsleading to monocytes recruitment within the tumor is thus a major issue in anti-cancertherapeutic protocols optimization. Based on the differential expression of chemokinereceptors CCR2 and CX3CR1, two populations of monocytes, inflammatory monocytes(CCR2+, CX3CR1low) and resident monocytes (CCR2-, CX3CR1high) have been characterized inmice which are involved in monocytes recruitment and differentiation into macrophages.The main objective of my work was to better understand the mechanisms of monocytesmobilization following chemotherapeutic treatment. I started to study the role of chemokinereceptors with a focus on CX3CR1 in monocytes reconstitution following cyclophosphamide(CP) treatment. CP is an alkylant agent known for its myelosuppressive properties. Thischemotherapeutic agent induces a transitory anti tumour immune response associated witha monocyte renewal and a strong infiltration of the tumour by adoptively transferred Tlymphocytes. However, antigen-specific T cells are trapped by tumour-associated dendriticcells (TuDCs). This potentially decreases interactions between T lymphocytes and tumourcells suggesting an immunosuppressive role of TuDCs. CP induces a strong depletion ofmyeloid cells followed by a massive reconstitution of bone marrow and spleen monocytesreservoirs. CX3CR1 expression on bone marrow monocytes is decreased duringreconstitution and correlated with a decreasing adhesion of these cells to CX3CL1 ex vivo.We highlighted an increased mobilization of inflammatory monocytes in CX3CR1-/- micecompared to WT and CCR2-/-. Intra vital imaging of bone marrow within CX3CR1-/- mice orwith the help of a CX3CR1 antagonist allowed us to show a specific role of CX3CR1 in thelumen crawling and confinement of monocyte-derived cells in both sinusoid andparenchyma of the bone marrow. We suggest that CX3CR1 controls the release of bonemarrow monocytes during CP-induced mobilization. We think that modulating the rate ofcellular mobilization, by increasing the host’s leukocyte pool during CP inducedreconstitution and/or targeting CX3CR1, could contribute to improve cellular responsefollowing tissue damage or immune cell dysfunction. Furthermore, targeting CX3CR1couldprovide applications in the stem cell transplantation domain.

The biological basis of the clinical heterogeneity in Parkinson's Disease (PD) is unclear. It is likely to involve complex interactions between genetic and environmental factors and between a range of pathological processes, including protein homeostasis and immune system function. Microglial activation in the brain and peripheral innate immune changes are known to occur in PD. Recently genetic, animal and cellular studies have linked several innate immune related genes and proteins (e.g. HLADR, TREM2, TLR2, TLR4, caspase-1) to PD and provided evidence that they may have a role in PD pathogenesis. Alpha-synuclein is central to PD, with evidence from neuropathology, genetics and animal/cell models indicating that it plays a significant pathogenic role. There is developing evidence directly linking innate immune activity and alpha-synuclein pathology. For example, inflammation, particularly in response to microbial infection, is associated with increased alpha-synuclein accumulation in the periphery and activation of the innate immune inflammasome related caspase-1 leads to increased cleavage and aggregation of alpha-synuclein. Overall Hypothesis- "Parkinson's disease (PD) and its clinical heterogeneity are associated with systemic changes in innate immune and associated microbial factors and in alpha-synuclein". This was investigated from the perspective of an epidemiological study, a study of peripheral blood monocyte, innate immune/microbial markers and a cerebrospinal fluid (CSF) study in PD patients. *The epidemiological study, involved the longitudinal PICNICS cohort of 290 Idiopathic PD patients, and showed that the use of medication known to influence alpha-synuclein and immune function is associated with motor heterogeneity in PD. *The peripheral immune study involved 41 early PD patients and 41 age, gender and MAPT genotype matched paired controls, with the PD patients categorised into 2 groups based on the presence of previously identified clinical and genetic risk factors for the development of an early dementia (impaired semantic fluency, pentagon copying and MAPT H1/H1 haplotype). This study demonstrated that the phenotypic profile of peripheral monocytes and the level of serum alpha-synuclein and relevant innate immune and microbial markers do differ in early PD compared to controls and that there are differential changes in those patients at higher versus lower risk for early dementia. The systemic alpha-synuclein related changes appear to be present overall in PD patients compared to controls, while the more microbial/innate immune related changes appear to be more prominent in the dementia higher risk group. *The CSF study involved samples from 35 PD patients and has demonstrated evidence of relationships between neurodegeneration-linked CSF tau species and inflammatory cytokines, and between CSF alpha-synuclein and cognitive function, suggesting that these factors may be involved in PD heterogeneity within the central nervous system as well. Overall, these studies provide evidence that variations in alpha synuclein/ tau homeostasis and innate immune and microbial factors are related to PD and its clinical heterogeneity.

La maladie de Parkinson (MP) est marquée par la présence d'une inflammation, pouvant être bénéfique ou délétère à la neurodégénérescence dopaminergique (DAgique). Nous avons adressé la nature des interactions pathologiques entre neurones DAgiques, cellules gliales et leucocytes infiltrant, nécessaire à la mise en place de cette inflammation. Dans un modèle MPTP murin de la MP, les objectifs de ma thèse ont été 1) d'identifier des signaux inflammatoires neuronaux et gliaux, par une approche transcriptomique associée à de la microdissection laser et 2) de déterminer leurs rôles dans la neuroinflammation ainsi que leurs effets sur la perte DAgique. Nous avons retenu parmi les candidats identifiés: les axes CXCL16-CXCR6 et CCL2-CCR2. Nous reportons dans le modèle MPTP, une expression microgliale de CXCL16 ainsi qu'une infiltration de population lymphocytaire CXCR6. Bien que la déplétion de CXCR6 permette de réduire cette infiltration, aucun effet n'est observé sur la perte DAgique. Nous décrivons une infiltration de monocyte CCR2 en concomitance avec une expression astrocytaire précoce de CCL2 dans le modèle MPTP murin, ainsi qu'une expression plus prolongée de CCL2 chez le primate non-humain MPTP, suggérant une relevance de l'axe CCL2-CCR2 dans la MP. En effet nous montrons que des souris surexprimant CCL2, intoxiquées au MPTP, ont non seulement une augmentation accrue de l'infiltrat monocytaire CCR2, mais également de la lésion DAgique. De manière inattendue, nous montrons que la neurotoxicité accrue observée chez des souris CX3CR1-/- MPTP passe indirectement par la voie CCL2-CCR2. Ainsi, nos données supportent l'hypothèse d'une neurotoxicité des monocytes CCR2 dans la MP. / Parkinson's disease (PD) presents signs of neuroinflammation, which can be beneficial or deleterious for dopaminergic (DA) neurodegeneration. We have analyzed the characteristics of such pathological interactions between DA neurons, glial cells and infiltrating immune cells. Using the neurotoxic MPTP mouse model of PD and focusing on chemokines, my thesis objectives were: 1) to identify using laser-microdissection and RNA profiling, the neuronal and glial inflammatory signals in the affected Substantia Nigra (SN) and 2) to assess the role of promising identified chemokine candidates during DA neurodegeneration. We have focused on the lymphocytic CXCL16-CXCR6 and the monocytic CCL2-CCR2 axes. We have found early microglial CXCL16 induction and parallel SN infiltration of CXCR6 lymphocyte subpopulations. CXCR6-deletion reduced infiltration of specific lymphocyte subpopulations, but did not affect the known deleterious infiltration of CD4 T-lymphocytes. For the CCL2-CCR2 axis, we found evidence for limited SN infiltration of CCR2 monocytes, which was preceded by transient astrocytic CCL2 induction in MPTP mice, but a prolonged CCL2 induction in MPTP monkeys, suggesting a potential relevance for human PD. While CCR2-gene deletion did not affect loss of DA neurons, astrocytic CCL2 overexpression increased MPTP induced DA neural loss, revealing the principally neurotoxic nature of infiltrating CCR2 monocytes in a PD-like environment. Unexpectedly, we also found that the known increased DA loss in CX3CR1-KO mice was mediated indirectly via over-induction of the CCL2-CCR2 axis. Combined, our results suggest a potential deleterious role of the CCL2-CCR2 axis in actual human PD.

Myocardial infarction (MI) is often caused by a disruption of an atherosclerotic plaque with activation of coagulation, platelets and inflammation. The aims were; to investigate whether the oral direct thrombin inhibitor, ximelagatran affected markers for coagulation, platelet and inflammation in a patient cohort with recent MI and if the coagulation markers could identify patients with increased risk of new ischemic events; to evaluate some of the mechanisms involved in formation of platelet-monocyte aggregates (PMAs). In a biomarker substudy patients with recent MI were randomized to 24-60 mg of ximelagatran or placebo for six months. There was a persistent dose-independent reduction of coagulation markers (F1+2, D-dimer) by ximelagatran treatment. 60 % reduced their D-dimer levels after one week and that group had less ischemic events during treatment. There was an early increase of the platelet activation marker and ximelagatran in higher doses attenuated these increased levels. Both in vivo and in vitro the direct thrombin inhibitor diminished procoagulant activity and tissue factor (TF) presenting microparticles. In contrast, the inflammatory markers increased after six months of ximelagatran treatment. The PMA-levels were elevated for long-term after MI. In vitro thrombin inhibition diminished formation of PMAs. Formation of PMAs in stimulated whole blood was P-selectin dependent and induced TF expression through phosphorylation of the Src-family member Lyn in monocytes. Addition of an oral direct thrombin inhibitor reduces coagulation and platelet activation markers for long-term after a MI together with reduced procoagulant activity which may contribute to the clinical benefit of the drug. Early reduction of D-dimer levels seems to be suitable to identify patients with reduced risk of new ischemic events independent of antithrombotic treatment. Circulating PMAs persist after a MI connecting coagulation to inflammation. Within these aggregates P-selectin induces TF, the main initiator of coagulation, partly through phosphorylation of Lyn.

Background: Patients with burns are susceptible to organ failure, and there is indirect evidence that leukocytes may contribute to this process. They may change the expression of cell-surface receptors after certain stimuli, for example, the burn. We therefore aimed to assess the changes induced by the burn in the expression of leukocyte cell-surface receptors CD11b, CD14, CD16, and CD62L on the surface of PMNs and monocytes. We also wanted to examine the dynamics of this activation during the first week after the burn, and to relate it to the size of the injury. Methods: Ten patients with burns of andgt;15% (TBSA) were included in the study. Blood samples were collected on arrival and every consecutive morning during the first week. Healthy volunteers acted as controls. Results: PMN CD11b expression was increased. The extent of PMN CD11b expression correlated negatively to the size of the full thickness burn. Monocyte CD14 expression increased initially but there was no relation to the size of the burn. PMN CD16 expression decreased initially during the first days and the decrease was related to burn size. CD62L did not vary depending on the burn in either PMN or monocytes during the first week after the burn. Conclusion: This study showed that specific receptors on the surface of leukocytes (PMN CD11b, monocyte CD14 and PMN CD16) are affected by the burn. Expression of PMN CD11b and CD16 are related to burn size. Burn-induced effects on the expression of PMN receptors, such as PMN CD11b and CD16, may contribute to burn-induced infection susceptibility. / Original Publication: Joakim Johansson, Florence Sjögren, Mikael Bodelsson and Folke Sjöberg, Dynamics of leukocyte receptors after severe burns: An exploratory study, 2011, BURNS, (37), 2, 227-233. http://dx.doi.org/10.1016/j.burns.2010.08.015 Copyright: Elsevier Science B.V., Amsterdam. http://www.elsevier.com/

A degradable/polar/hydrophobic/ionic (D-PHI) polyurethane with properties intended to promote tissue regeneration in a small diameter peripheral artery vascular graft was evaluated for cell biocompatibility and growth. Films were cast in polypropylene 96 well plates for monocyte/endothelial cell (EC) co-culture in vitro studies and porous scaffold discs were implanted in an in vivo subcutaneous mouse model. After 7 days in culture the co-culture demonstrated cell adhesion and growth, low esterase activity (a measure of degradative potential and cell activation), no detectable release of pro-inflammatory cytokine (tumour necrosis factor -α) but measurable anti-inflammatory interleukin (IL)-10. The EC and the co-culture expressed the EC biomarker CD31, whereas the monocyte monoculture did not. Cytokine array analysis of the in vivo characterization of D-PH supported an anti-inflammatory phenotype of cells at the site of the implant. Levels of IL-6 significantly decreased over time while IL-10 was significantly higher at 6 weeks post implant. TNF-α levels did not change significantly from 24 hours onwards, however the trend was towards lesser amounts following the initial time point. Histological analysis of the explanted scaffolds showed excellent tissue ingrowth and vascularization. A live/dead stain showed that the cells infiltrating the scaffolds were viable. Both the in vitro and in vivo results of this thesis indicate that D-PHI is a good candidate material for tissue engineering a peripheral artery vascular graft.