Macrophage migration, an essential component of many biological processes and pathologic conditions, is mediated by integrated cellular signaling processes and cytoskeleton rearrangement. Recent advances indicate that the innate immunity signaling process plays a key role in the regulation of macrophage migration. Furthermore, our lab has provided evidence demonstrating the involvement of a key innate immunity signaling kinase, IRAK-1, as a critical modulator of murine macrophage migration. Macrophage migration induced by a potent PKC activator, phorbol 12-myristate 13-acetate (PMA), or lipopolysaccharide (LPS) was significantly decreased in IRAK-1-/- murine macrophages compared with wild type cells. Mechanistically, we first demonstrated that IRAK-1 works downstream of PKCε and directly binds to VASP, a cytoskeleton regulatory protein, to regulate PMA-induced macrophage migration. Secondly, we proved that IRAK-1 is required for LPS-induced macrophage migration and expression of MCP-1, a chemotactic cytokine for macrophages, via transcription factor C/EBPδ instead of NFκB. IRAK-1 binds directly to IKKε and inhibition or knock-down of IKKε results in a significant decrease in C/EBPδ expression and MCP-1 mRNA expression. Lastly, we identified the direct association between IRAK-1 and Rac1, a member of the Rac subfamily in the Rho family of GTPases. These finding further confirmed the essential role of IRAK-1 during macrophage migration. Our research provides a novel facet regarding the molecular signaling processes regulating macrophage migration. / Ph. D.

Osteoarthritis (OA) is the leading cause of musculoskeletal disability in people and horses, and is characterized by progressive joint degeneration. There is a critical need for a better understanding of disease processes leading to OA in order to develop more efficient therapies. A shared feature among different arthritic conditions is chronic synovitis. Macrophages are the main drivers of synovitis and can display pro-inflammatory (M1) or pro-resolving responses (M2). Macrophages promote joint health through phagocytic and secretory activities; however, when these functions are overwhelmed, macrophages upregulate inflammation, recruiting more cells to counteract damage. Once cell recruitment is efficiently accomplished, macrophages coordinate tissue repair and further resolution of inflammation. Bone marrow mononuclear cells (BMNC) are a source of macrophages used to treat inflammation and produce essential molecules for cartilage metabolism; however, little information exists regarding their use in joints. The studies presented in this dissertation focus on understanding the dual role of macrophages in driving and resolving synovitis and how to harness their therapeutic potential. In the first study, patterns of macrophage phenotypes (M1:M2) in healthy and osteoarthritic equine synovium were compared and correlated with gross pathology, histology, and synovial fluid cytokines. M1 and M2 markers were co-expressed in normal and osteoarthritic joints, varying in intensity of expression according to degree of inflammation. Concentrations of synovial fluid IL-10, a macrophage-produced cytokine that is vital for chondrocyte recovery from injury, was lower in OA joints. The combined findings of this study suggest homeostatic mechanisms from synovial macrophages in OA may be overwhelmed, preventing inflammation resolution. In the second study we investigated the response of BMNC to normal (SF) and inflamed synovial fluid (ISF). BMNC cultured in autologous SF or ISF developed into macrophage cultures that were more confluent in ISF (~100%) than SF (~25%), and exhibited phenotypes that were ultimately similar to cells native to normal joints. BMNC cultured in SF or ISF were neither M1 nor M2, but exhibited aspects of both phenotypes and a regulatory response, characterized by increasing counts of IL-10+ macrophages, decreasing concentrations of IL-1β, and progressively increasing concentrations of IL-10 and IGF-1, all more marked in ISF. These findings suggest that homeostatic mechanisms were preserved over time, and potentially favored by macrophage proliferation. Our data suggest that BMNC therapy could potentiate the macrophage- and IL-10-associated mechanisms of joint homeostasis lost in OA. Finally, using an equine model of synovitis, the last study investigated the response of normal and inflamed joints to autologous BMNC injection. Inflamed joints treated with BMNC showed gross and analytical improvements in synovial fluid and synovial membrane, with increasing numbers of regulatory macrophages and synovial fluid concentrations of IL-10, not observed in saline-treated controls. Autologous BMNC are readily available, downregulate synovitis through macrophage-associated effects, and can benefit thousands of patients with OA. Combined, the results of these studies support the role of macrophage-driven synovial homeostasis and identified a therapeutic way to recover homeostatic mechanisms of synovial macrophages lost during chronic inflammation. Our findings also uncover new research directions and methods for future studies targeting modulation of joint inflammation. / Doctor of Philosophy / Osteoarthritis (OA) is a common cause of joint deterioration in people and horses. Current treatments provide limited recovery of joint function, creating an urgent need for more efficient therapies; however, development of new treatments requires better understanding of the mechanism causing OA. A shared characteristic among many arthritic conditions is long-standing inflammation. Cells called macrophages are the main drivers of joint inflammation and can exert pro- and anti-inflammatory effects. Macrophages promote joint health by clearing aggressor agents and secreting molecules required for optimal joint function. However, when these housekeeping functions are overwhelmed by damage, macrophages drive inflammation recruiting more cells to cope with increased demands for repair. If this process is efficiently accomplished, macrophages then resolve inflammation, recovering joint health. Macrophages in the bone marrow (BMNC - bone marrow mononuclear cells) are used to treat inflammation in several tissues and are known to produce molecules essential for joint health. Although little information exists regarding their use in joints, studies treating different organs suggest it can provide high rewards. The studies presented in this dissertation focused on understanding the dual function of macrophages in driving and controlling joint inflammation, and harnessed their therapeutic potential. In the first study, macrophages were investigated in normal and OA-affected joints, and curiously exhibited a hybrid pro- and anti-inflammatory identity in both groups. The indicators of this mixed identity were more markedly expressed in arthritic joints showing gross inflammation. Low levels of a macrophage-derived anti-inflammatory protein called IL-10 were detected in OA joints. The results of this study suggest that anti-inflammatory mechanisms from macrophages may be overwhelmed in OA-affected joints, preventing inflammation to be resolved, and that recovering this anti-inflammatory function may aid in the treatment of OA. In the second study we investigated how the incubation of BMNC in fluid from normal and inflamed joints affects the response of macrophages. Similar to what we observed in the first study, BMNC incubated in both normal and inflamed joint fluid induced macrophages to develop a hybrid identity that was ultimately similar to native cells from normal joints. Macrophages proliferated more when incubated in fluid from inflamed joints. Macrophages in both groups produced anti-inflammatory effects with high levels of IL-10 that were highest in ISF cultures. These observations suggest that higher proliferation of macrophages in inflamed joint fluid helped preserve anti-inflammatory mechanisms. Therefore, our study suggests that joint injection with BMNC could maximize macrophage- and IL-10-associated mechanisms required to resolve joint inflammation. The third and final study investigated the response of normal and inflamed joints to BMNC injection using a model of joint inflammation in horses. Inflamed joints treated with BMNC showed visual and laboratorial markers of improvement, with increasing numbers of macrophages and concentrations of IL-10 in the joint fluid, which remained lower in joints treated with placebo. BMNC provide means to recover macrophage-associated effects required to control joint inflammation and can benefit thousands of patients with OA. Together, the results of these studies show that macrophages are biased promoters of joint health, leading to inflammation when their anti-inflammatory mechanisms are overwhelmed. Replenishing inflamed joints with healthy macrophages maximizes their anti-inflammatory effects, favoring the recovery of a healthy articular environment.

Chez les métazoaires, la différenciation terminale est généralement accompagnée par une sortie définitive du cycle cellulaire. Cependant, les macrophages et très peu d'autres types cellulaires rompent avec ce dogme. En effet, il est maintenant admis que les macrophages conservent la capacité de s'auto-renouveler indépendamment des cellules souches ou progénitrices. À cet égard, nous avons démontré que la double déficience en facteurs Maf dans les macrophages (Maf-DKO) leur confère la capacité de s'auto-renouveler indéfiniment en culture sans se dé-différencier ou devenir tumorigènes. Ce phénotype d'auto-renouvellement semble être médié par un réseau transcriptionnel de de gènes régissant l'auto-renouvellement qui sont également actifs dans les cellules souches embryonnaires, parmi lesquels Myc et Klf4. Ces deux facteurs sont activés et nécessaires pour l'auto-renouvellement des Maf-DKO. De façon intéressante, l'expression de Myc seul induit une prolifération illimitée des macrophages, mais provoque une transformation tumorale. Nous avons donc cherché à décrypter les mécanismes grâce auxquels Myc et Klf4 induisent l'auto-renouvellement des macrophages, en comparaison à la transformation cellulaire causée par l'expression de Myc uniquement. En outre, nous nous sommes concentrés sur l'identification de gènes candidats permettant un auto-renouvellement illimité des macrophages, tout en les protégeant de la transformation cancéreuse. Notre objectif est de contribuer à l'identification du programme transcriptionnel régulant l'auto-renouvellement non tumoral des macrophages. / In metazoan, terminal differentiation is generally accompanied by permanent exit from the cell cycle. Yet, macrophages and very few other examples break with this dogma. Indeed, it has become evident that macrophages retain the ability to self-renew independently of stem or progenitor cells. In this regard, we have previously shown that MafB/c-Maf double deficient (Maf-DKO) macrophages are able to self-renew indefinitely in vitro without dedifferentiating or becoming tumorigenic. This self-renewal phenotype appears to be mediated by a transcriptional network of self-renewal genes also active in embryonic stem cells, among which Myc and Klf4. Interestingly, these two factors are activated and required for Maf-DKO self-renewal. By contrast, Myc alone induces an unlimited proliferation of macrophages but causes malignant transformation. We aimed to decipher the mechanisms by which Myc and Klf4 induce stem cell-like self-renewal in macrophages, in comparison to cellular transformation caused by the expression of Myc alone. Additionally, we focused on identifying candidate genes allowing an unlimited self-renewal of macrophages while protecting them from tumorigenic transformation or aberrant proliferation. Our objective is to contribute to the identification of the transcriptional program regulating non-tumorigenic self-renewal in macrophages.

Selon les facteurs solubles de l’environnement (cytokines), les monocytes peuvent se différencier en macrophages (Mφ) ou en cellules dendritiques. Mes travaux de thèse montrent que l’IL-34, un second ligand du M-CSF-R (c-fms ou CD115), induit la différenciation de monocytes humains en macrophages CD14+ CD163+ (IL-34-Mφ), phénotypiquement et fonctionnellement similaires aux M-CSF-Mφ et aux macrophages associés aux tumeurs (TAM) isolés du cancer de l’ovaire. Ils possèdent des propriétés immunosuppressives et diminuent la prolifération des lymphocytes T CD4+ activés. Comme les Mφ orchestrent la réponse immunitaire, j’ai évalué la capacité des M-CSF-Mφ, IL-34-Mφ et des TAM à polariser les LT CD4+ mémoires. Les résultats montrent qu’ils polarisent des LT non-Th17 mémoires en LTh17 CCR4+ CCR6+ CD161+ conventionnels exprimant ou non de l’IFN. Ce processus est médié par l’expression constitutive de l’IL-1α membranaire exprimées sur ces macrophages.Dans le but d’identifier des stratégies pour prévenir l’accumulation de ces Mφ dans le cancer, j’ai montré que (i) l’IFNγ et le GM-CSF inhibent la différenciation des M-CSF-Mφ et IL-34-Mφ et que (ii) l’IFNγ permet la réversion des M-CSF-Mφ et IL-34-Mφ en Mφ immunostimulants. En conclusion, mes travaux montrent que les M-CSF-Mφ, IL-34-Mφ et les TAM, considérés initialement comme anti-inflammatoires, induisent la génération de lymphocytes Th17 via une expression constitutive de l’IL-1α. Ce processus pourrait contribuer à maintenir, localement, une inflammation modérée nécessaire au développement tumoral. / According to the soluble factors in the environment (cytokines), monocytes differentiate into macrophages (Mϕ) or dendritic cells. This project demonstrates that IL-34, a second ligand of the M-CSF receptor (c-fms or CD115), induces the differentiation of human monocytes into CD14high CD163 high Mφ (IL-34-Mφ), phenotypically and functionally similar to M-CSF-Mφ and to tumorassociated macrophages (TAM) isolated from the ovarian cancer. They exhibit potent immunosuppressive properties and decrease the proliferation of stimulated Tcells. As Mφ orchestrate the immune response, I have evaluated the capacity of M-CSF-Mφ, IL-34-Mφ and TAM to polarize human memory CD4+ T cells. Unexpectedly, results showed that they switch non- Th17 memory CD4+ T cells into conventional CCR4+ CCR6+ CD161+ Th17 cells, expressing or not IFNγ. This process is mediated by the constitutive expression of membrane IL-1α on these Mϕ subsets. In an attempt to identify strategies to prevent an accumulation of immunomodulatory Mϕ in cancer, I have shown that (i) IFNγ and GM-CSF prevent M-CSFandIL-34-induced monocyte differentiation into immunosuppressive Mφ and (ii) that IFNγ switchesestablished M-CSF-Mφ and IL-34-Mφ into immunostimulatory Mφ. In conclusion, this study demonstrates that human MCSF- Mφ, IL-34-Mφ and TAM initially considered as antiinflammatorycells, induce in vitro Th17 cell generation via a constitutive expression of membrane IL-1α. This process may contribute to maintain locally a restrained and smoldering inflammation required for angiogenesis and metastasis in tumors.

Post-operative ileus (POI) is the functional inhibition of propulsive intestinal motility which is a frequent occurrence following abdominal surgery in the horse and in humans. Rodent and human-derived data have shown that manipulation-induced activation of the resident muscularis externa (ME) macrophages in the intestine contributes to the pathophysiology of the disease. Most studies of the disease, specifically in the horse, have focussed on identification of risk factors, descriptive studies of the disease or the assessment of the efficacy of various therapeutic and prophylactic interventions. As a result, the proposed pathogenesis of equine POI is largely reliant on the translation of data from rodent models. The aims of this thesis were to identify macrophage populations in the normal equine gastrointestinal tract (GIT) and to study equine macrophage activation by stimulating equine bone marrow-derived macrophages (eqBMDMs) with lipopolysaccharide (LPS) as a model for intestinal macrophage activation. Firstly, the normal population of macrophages in the equine GIT was determined. Using CD163 as an immunohistochemical marker for macrophages. CD163+ve cells were present in all tissue layers of the equine intestine: mucosa, submucosa, ME and serosa. CD163+ve cells were regularly distributed within the ME, with accumulations adjacent to the myenteric plexus, and therefore to intestinal motility effector cells such as neurons and the Interstitial Cells of Cajal. The differentiation and survival of intestinal macrophages depends upon signals from the macrophage colony-stimulating factor (CSF-1) receptor. LPS translocation from the gut lumen is thought to be a key activator of ME macrophages. To provide a model for gut macrophages, a protocol was optimised to produce pure populations of equine bone marrow-derived macrophages (eqBMDMs) by cultivation of equine bone marrow in CSF-1. Macrophage functionality was assessed using microscopy, flow cytometry and phagocytosis assays. EqBMDMs responded to LPS stimulation with increases in expression of positive control genes, tumour necrosis factor alpha (TNF-α) and Indoleamine 2,3-dioxygenase (IDO1). The same mRNA was subjected to transcriptomic (RNA-Seq) analysis. Differential gene expression and network cluster analysis demonstrated an inflammatory response characterised by the production of pro-inflammatory cytokines such as interleukin 1 beta (IL-1β) and interleukin 6 (IL-6). However, in contrast to rodent macrophages, eqBMDMs failed to produce nitric oxide in response to LPS, showing species-specific variation in innate immune biology. Using these data, we compared gene expression in normal equine intestine and in intestine from horses undergoing abdominal surgery for colic (abdominal pain). Horses undergoing abdominal surgery showed evidence of increased expression of IL-1β, IL-6 and TNF-α in the mucosa and ME when compared to control tissue. Horses with post-operative reflux (POR), a clinical sign of POI, had increased gene expression of IL-1β, IL-6 and TNF-α compared to horses that did not develop POR following abdominal surgery. These preliminary data suggest that there is macrophage activation within the ME of the intestine during abdominal surgery in the horse, and that a greater activation state is present in horses that subsequently develop POR. The final part of this study was to investigate the effect of a long-acting form of CSF- 1, an Fc fusion protein (CSF1-Fc), as a potential treatment for POI using a mouse model. This work, performed in collaboration with another research group, found that mice lacking the C-C chemokine receptor type 2 (CCR2) gene, which is required for monocyte recruitment into tissues, had a longer recovery period following intestinal manipulation (IM) than wild type (WT) mice. With the administration of CSF1-Fc, infiltration of neutrophils to the ME was reduced and the number of macrophages in the ME was increased in both WT and CCR2-/- mice following IM. Administration of CSF1-Fc in CCR2-/- mice improved recovery of gastrointestinal transit three days following IM, to the same extent as WT mice. Network cluster analysis and RT-qPCR of the ME revealed clusters of genes induced and downregulated by CSF1-Fc, with increased expression of anti-inflammatory and pro-resolving genes after IM in WT and CCR2-/- mice following treatment with CSF1-Fc.

Macrophage-derived inflammation contributes to chronic inflammation in adipose tissue in obesity and is also linked to the development of skeletal muscle (SM) insulin resistance. The long-chain n-3 PUFA have been shown to modulate cytokine secretion from macrophages, though subsequent effects on SM inflammation and function are unknown. A model of macrophage conditioned media (MCM) was used to examine effects of n-3 PUFA on macrophage inflammation and consequent effects on SM cells. Treatment of RAW 264.7 macrophages with long-chain n-3 PUFA decreased LPS-induced MCP-1 and IL-6 gene expression and MCP-1 secreted protein. In turn, MCM from n-3 PUFA-treated macrophages decreased TNF-α and IL-6 gene expression in LPS-stimulated L6 SM cells, but did not affect insulin-stimulated pAkt content. Long-chain n-3 PUFA did not affect gene expression of inflammatory signaling intermediates NF-κB and TLR4. Overall this thesis suggests that long-chain n-3 PUFA are important nutritional strategies for reducing macrophage-derived inflammation, with ensuing benefits in SM inflammation. / NSERC-CGS, Ontario Graduate Scholarship

During our lifetime, our bones constantly go through remodelling to maintain the skeletal system. This is done by osteoblasts that deposit new bone tissue, osteoclasts that remove the bone matrix and mechanosensing osteocytes. In case of bone implants, increased resorption by osteoclasts due to inflammation (inflammatory osteolysis) leads to aseptic implant loosening. This study focuses on how to detect these inflammatory resorbing cells at an early stage and prevent their activity with appropriate medication. To achieve this, we differentiated classical monocytes into macrophage-like cells, osteoclasts(OCs) and foreign body giant cells (FBGC) and their secretome was studied to identify specific biomarkers. Previously, tartrate resistant acid phosphatase (TRAP) was studied as an important biomarker for OCs and macrophages. An ELISA to separate and quantitate the two TRAP isoforms was used to distinguish the resorbing OCs from inflammatory FBGCs on the basis of the isoform ratio. This assay gave high levels of 5b isoform for osteoclastic stimulation and high 5a levels for the inflammatory stimulation. Also, different aminothiazole inhibitors were tested which were shown to be efficient drugs in inhibiting inflammatory osteolysis by reducing osteoclast formation and resorption in sub-micromolar concentration. Further to apply this study to patient samples, an immunoassay panel can be developed which will help detect TRAP and multiple biomarkers like CTX specific to aseptic loosening simultaneously. This will help in early, efficient and accurate diagnosis of inflammatory osteolytic bone loss and provide us with an accurate diagnosis and sufficient time for appropriate treatment.

Protozoa of the Leishmania genus are transmitted between mammalian hosts by the sandfly and cause the neglected tropical disease leishmaniasis. Upon injection into the mammalian host by the sandfly promastigote-form parasites are phagocytosed by macrophages, where they differentiate into amastigotes. Although many virulence factors are known to modulate macrophage signalling pathways to favour infection, the delivery mechanisms are largely unknown. During differentiation to amastigotes the promastigote flagellum shortens dramatically and the fate of the excess flagellar membrane is unknown. Here we investigate the possibility that during Leishmania mexicana differentiation, shedding of the flagellar membrane is a source of extracellular vesicles (EVs) which provide a virulence factor delivery mechanism. The kinetics and structural mechanisms of EV release from promastigotes were investigated by live cell imaging and by measuring the concentration of shed EVs. Isolated EVs from a differentiating parasite culture or a control promastigote parasite culture were analysed by fluorescence and electron microscopy and mass spectrometry. To study the biological effects of EVs, macrophages were exposed to isolated EVs or live promastigotes and cytokine secretion was quantified by ELISA. An LPG1 null mutant was used to assess the contribution of virulence factor lipophosphoglycan (LPG) to the observed effects. Known protein virulence factors and LPG are present in L. mexicana EV fractions as well as known flagellar proteins. We show that there is a link between L. mexicana flagellar shortening and EV release, which is a recently discovered phenomenon in Chlamydomonas and mammalian cell research. We find that isolated EVs and live promastigotes induce changes in secreted cytokine levels from human and murine macrophages, including a substantial and previously unreported suppression of CXCL10, a chemokine which plays a protective role in Leishmania infection. LPG contributes to the effects observed on cytokine production, and EVs may be an important delivery mechanism for LPG.

L'infiltration des mélanomes par les macrophages (TAM) est souvent corrélée à un mauvais pronostic. Cependant, les mécanismes qui régulent le recrutement et la fonction de ces cellules restent encore mal compris. Des études récentes ont montré un rôle majeur de la sphingosine kinase 1 (SK1) tumorale, l'enzyme qui produit la sphingosine-1-phosphate (S1P), dans le remodelage du stroma associé à la tumeur. Le but de ce projet a été d'étudier le rôle de la SK1 tumorale sur le microenvironnement inflammatoire, et en particulier les macrophages, lors du développement des tumeurs mélaniques. In vitro, nous avons montré que l'inhibition de SK1 dans les cellules de mélanome : 1) bloque la migration des macrophages. A l'inverse, la surexpression de cette kinase dans les cellules tumorales stimule la migration des cellules inflammatoires. Cet effet est dépendant de la S1P et de sa fixation sur les récepteurs S1PR présents à la surface des macrophages ; 2) réduit la sécrétion de TGF-ß et 3) stimule la différenciation des macrophages vers un phénotype M1 antitumoral. Ce phénomène n'est pas dépendant de la S1P, ni des S1PRs, mais de la sécrétion de TGF-ß par les cellules tumorales. En effet, la différenciation macrophagique peut-être réversée par l'addition de TGF-ß recombinant dans le milieu de sécrétion des cellules tumorales. In vivo, nos résultats montrent que la greffe orthotopique, i.e. intradermique, de cellules de mélanome murin invalidées pour la SK1 à des souris syngéniques C57BL/6 est associée à une réduction de la croissance tumorale, comparée à des souris ayant reçu des cellules de mélanome contrôles. De plus, l'invalidation de la SK1 tumorale conduit à une augmentation significative de l'expression de cytokines anti-tumorales ainsi qu'à une polarisation Th1 au sein de la tumeur. Ce phénomène s'accompagne d'une réduction du pourcentage de macrophages M2 CD206+MHCIIlow, et à l'inverse, d'une augmentation du pourcentage de macrophages M1 CD206-MHCIIhigh ainsi que de lymphocytes T CD4+ et CD8+ infiltrés dans la tumeur. Ces résultats suggèrent un rôle clé de la SK1 tumorale dans le recrutement et la polarisation des macrophages dans les mélanomes. Ainsi, l'axe SK1/ TGF-ß pourrait constituer une cible thérapeutique prometteuse dans le contrôle de la croissance de cette tumeur. / Melanoma infiltration by macrophages (TAM) is often correlated with poor prognosis. However, the mechanisms that regulate the recruitment and function of these cells remain poorly understood. Recent studies have shown a major role of tumor sphingosine kinase 1 (SK1), the enzyme that produces sphingosine-1-phosphate (S1P), in tumor stroma remodeling. The aim of this project was to investigate the role of tumor SK1 on the inflammatory microenvironment, particularly macrophages, during the development of melanoma. In vitro, we showed that the inhibition of SK1 in melanoma cells: 1) blocks macrophage migration. Conversely, overexpression of this kinase in tumor cells stimulates the migration of inflammatory cells. This effect is dependent on S1P binding to its receptors (S1PR) on the macrophage surface; 2) reduces the secretion of TGF-ß and 3) stimulates macrophage differentiation towards an antitumor M1 phenotype. The latter phenomenon does not depend on S1P nor S1PRs, but on the secretion of TGF-ß by tumor cells. Indeed, macrophage differentiation can be reversed by adding recombinant TGF-ß in the tumor cell-conditioned medium. In vivo, our results showed that orthotopic injection, i.e. intradermal, of murine melanoma cells invalidated for SK1 in C57BL / 6 syngenic mice was associated with a reduction in tumor growth compared to mice having received control melanoma cells. Furthermore, the invalidation of tumor SK1 leads to a significant increase in the expression of anti-tumor cytokines and a Th1 polarization within the tumor. This phenomenon is accompanied by a reduction in the percentage of CD206+MHCIIlow M2 macrophages, and conversely, an increase in the percentage of M1 macrophages CD206-MHCIIhigh as well as CD4+ and CD8+ cells infiltrated into the tumor. These results suggest a key role of tumor SK1 in the recruitment of macrophages and their polarization in melanoma. Thus, the axis SK1 / TGF-ß could be a promising therapeutic target in controlling the growth of this tumor.

Les macrophages (MΦ) du système nerveux central forment la microglie, qui contrôle son homéostasie. Plusieurs modèles de "fate mapping" ont montré que la microglie provenait du Sac vitellin (SV). Celui-ci produit les MΦ en deux vagues indépendantes. Dans la première, des progéniteurs restreints génèrent des MΦ primitifs, alors que dans la seconde, des progéniteurs érythro-myéloïdes produisent des MΦ définitifs. Les progéniteures primitifs et définitifs ont les mêmes phenotype et voie de différenciation. Leurs spécificités et leurs contributions aux étapes ultérieures du développement sont donc encore incomprises. Nous montrons que l'expression du facteur de transcription Lyl-1 discrimine les populations de MΦ primitifs et définitifs. Les MΦ primitifs Lyl-1+ fournissent la microglie de l'embryon. De plus, l'invalidation de Lyl-1 disruption conduit à une production accrue de MΦ primitive dans le SV précoce, puis à une réduction du contingent microglial à deux stades spécifiques du développement. Lyl-1 est spécifiquement exprimé par la microglie et aucun autre type cellulaire nerveux. Son inactivation conduit à des modifications comportementales typiques de l'anxiété sociale. Nous identifions donc Lyl-1 comme un marqueur des MΦ primitifs du SV qui donnent naissance à la microglie de l'embryon. Nous montrons également que Lyl-1 contrôle l'expansion et la différenciation de la microglie, et est ainsi impliqué dans la régulation des processus du neuro-développement. / Microglia are tissue macrophages (MΦ) of the central nervous system that control tissue homeostasis. Different fate mapping models have shown that microglia originates from the yolk sac (YS). Macrophages production in the YS occurs in two independent waves. In the first, primitive MΦ originate from restricted progenitors, while in the second, definitive MΦ are produced by erythro-myeloid progenitors. Because primitive and definitive MΦ progenitors share the same phenotype and differentiation pathway, their specific features and contribution to further developmental steps are still poorly understood. We here show that the expression of thee transcription factor Lyl-1 discriminates primitive and definitive MΦ populations. YS-derived Lyl-1+ primitive MΦ contribute to embryonic microglia. Moreover, Lyl-1 disruption results in an increased production of primitive MΦ progenitors in the early YS. It also leads to the reduction of the microglia pool at two specific development stages. Lyl-1 is specifically expressed in microglia, but not other brain cells and its inactivation leads to behavioral changes typical for social anxiety disorders. Thus, we identify Lyl-1 as a marker for YS primitive MΦ that will give rise to the entire microglia. We show that Lyl-1 controls microglia expansion and differentiation and is involved in the regulation of neurodevelopmental processes.