Abstract Human microglia, monocyte-derived dendritic cells (DCs) and macrophages ex vivo express relatively higher levels of sphingosine-1-phosphate (S1P) receptor 1 (S1P1) mRNA as compared to other ...receptor subtypes. The S1P agonist FTY720 decreased ERK phosphorylation and induced myosin light chain (MLC) II phosphorylation only in macrophages and DCs. FTY720 inhibited IL-12p70 production (CD40L induced) by DCs and macrophages but not microglia (poly I:C induced). IL-10 production was increased in DCs and unaffected in other myeloid cells. Despite similar receptor expression patterns, the distinct myeloid cell populations present in the human CNS, under steady-state or inflammatory conditions, exhibit differential responses to FTY720.
FTY720 (fingolimod) treatment of multiple sclerosis (MS) results in lymphopenia due to increased recruitment into and decreased egress from secondary lymphoid organs of CCR7(+) lymphocytes. Although ...absolute numbers of NK lymphocytes were reported as being unaltered in FTY720-treated MS patients (MS-FTY), such analyses did not detect a change in a minor subset. Because expression of CCR7 has been described on CD56(bright) NK cells, a minority population of NK cells, we investigated the effect of FTY720 treatment on the phenotype and function of human NK cells in the peripheral circulation of MS patients. MS-FTY patients displayed a decreased proportion of peripheral CD56(bright)CD62L(+)CCR7(+) NK cells compared with untreated MS and healthy donors. In vitro treatment with FTY720-P increased migration of untreated donor NK cells to CXCL12 while reducing the response to CX3CL1 with similar migration responses seen in NK cells from MS-FTY patients. FTY720-P inhibited sphingosine 1-phosphate-directed migration of CD56(bright) and CD56(dim) NK cells subsets from untreated healthy donors. IL-12- and IL-15-stimulated NK cells from MS-FTY patients displayed similar capacity to produce IFN-γ, TNF, IL-10, and MIP-1α cytokines/chemokines compared with NK cells from untreated healthy donors and displayed comparable levels of degranulation in response to K562 tumor cells compared with untreated donors. Subset alterations and function of NK cell populations will need to be considered as part of assessing overall immunosurveillance capacity of patients with MS who will receive sustained FTY720 therapy.
Abstract The molecular mechanisms that underlie the axonal damage that accompanies CNS inflammation are largely unknown. Here, we investigate the effects of immune cells on neuronal viability and ...axonal growth and show that conditioned media from myeloid lineage cells inhibit neurite outgrowth without causing apoptosis. Treatment with monocyte conditioned medium enhances myosin light chain phosphorylation in neurons and the neurite outgrowth inhibitory effect of myeloid lineage cells can be attenuated with the myosin II inhibitor blebbistatin. Our results suggest that in the context of CNS inflammation myeloid cells may limit axonal repair in the CNS via a myosin II-dependent mechanism.
Microglia are an important component of the innate immune system within the central nervous system (CNS). Isolation and in vitro culturing of microglia can provide insight towards the basic biology ...of these cells as well as their interactions with neurons, astrocytes, and oligodendrocytes. While studies of rodent microglia and microglial cell lines have provided a basis for our understanding of these cells, human adult microglia exhibit distinct properties when compared to rodent cells. Furthermore, the study of human fetal microglia provides a window into the developing CNS. This chapter describes the protocols used to isolate, purify, and culture both human adult and fetal microglia. Under basal culture conditions, human microglia survive for extended periods in the absence of growth factors, thus allowing their properties to be investigated under resting conditions. In addition, both human adult and fetal microglia can be used to study how they respond to different polarization conditions. As is the case with macrophages, it is also possible to polarize microglia towards the pro-inflammatory "M1" and the anti-inflammatory "M2" phenotypes, as described in this chapter.
Measuring the effects of sphingosine-1-phosphate (S1P) receptor modulators on human primary neural cells is of particular interest given the recent application of these central nervous ...system-accessible agents to the treatment of neurodegenerative diseases, such as multiple sclerosis. Issues to consider in experimental studies include the ability of some of these modulators to bind multiple S1P receptor subtypes simultaneously, the nonspecificity of commercially available S1P receptor antibodies, and activation of multiple intracellular signaling cascades by a given S1P receptor. Here, we discuss how to assay S1P receptor expression and activation using multiple agonists/antagonists, by linking the results of real-time reverse transcriptase polymerase chain reaction with the assessment of intracellular signaling derived from Western blot analyses.
In the human central nervous system (CNS), the two major populations of myeloid cells are the blood-derived macrophages and the CNS-resident microglia. Under physiologic conditions, macrophages ...occupy the perivascular spaces and microglia populate the CNS parenchyma. Both cell types may be recruited to participate in neuroinflammatory responses such as in multiple sclerosis (MS) lesions, as macrophages access the parenchyma and the cell types become difficult to distinguish. Differential responses of macrophages and microglia to environmental conditions and to therapeutic agents could be important determinants of outcome in neuroinflammatory diseases of the CNS of which multiple sclerosis is the prototype. Experiments described in this thesis were based on use of microglia derived from the adult human CNS and myeloid cells generated from peripheral blood samples. Comparisons were made with regard to responses to the novel MS therapeutic FTY720, experimental polarizing conditions, and exposure to the CNS breakdown product myelin. As an indication of their propensity to respond to FTY720 treatment, the myeloid cell subsets were examined for their expression of sphingosine-1-phosphate (S1P) receptor mRNA. Human microglia, monocyte-derived dendritic cells (DCs) and macrophages ex vivo express relatively higher levels of sphingosine-1-phosphate (S1P) receptor 1 (S1P1) mRNA as compared to other receptor subtypes. Despite these similar receptor expression patterns, the S1P agonist FTY720 decreased extracellular-signal-regulated kinases (ERK) phosphorylation and induced myosin light chain (MLC) II phosphorylation in macrophages and DCs but not in microglia. FTY720 inhibited interleukin (IL)-12p70 production (CD40L induced) by DCs and macrophages but not microglia (poly I:C induced). IL-10 production was increased in DCs following FTY720 treatment and unaffected in the other myeloid cells. In terms of responses to polarizing conditions, macrophages can be polarized into a continuum of activation phenotypes, the extremes of which are the “pro-inflammatory” M1 and “anti-inflammatory” M2 phenotypes. These phenotypes have been linked to functional properties including production of inflammation association molecules and phagocytic activity. The phenotypic and functional properties of microglia were compared with macrophages derived from peripheral blood monocytes in response to M1 and M2 inducing conditions. Under M1 conditions, microglia and macrophages up-regulated expression of the M1 markers CCR7 and CD80. M2 treatment of microglia induced expression of CD209 but not the additional M2 markers CD23, CD163 and CD206 expressed by M2 macrophages. M1-polarizing conditions induced production of IL-12p40 by both microglia and macrophages; microglia produced higher levels of IL-10 under M1 conditions than did macrophages. Under M2 conditions, microglia produced comparable levels of IL-10 as under M1 conditions, whereas IL-10 was induced in M2 macrophages subsequently activated with lipopolysaccharide (LPS). Myelin phagocytosis was significantly greater by microglia than macrophages under all conditions; activity was higher for M2 treated cells for both cell types. Our findings delineate distinctive properties of microglia compared to exogenous myeloid cells both under physiological conditions and in response to signals derived from an inflammatory environment in the CNS. The results that myeloid cells exhibit differential responses to an approved MS therapeutic agent suggest that targeting the distinct myeloid populations and promoting M2 polarization, especially in the infiltrating macrophages, may be a potential therapeutic target to control inflammation in the CNS.
Les principales populations de cellules myeloïdes du système nerveux central (SNC) sont les macrophages, provenant de monocytes sanguins, et les microglies, résidant au sein du SNC. Les macrophages occupent normalement l'espace périvasculaire alors que les microglies peuplent le parenchyme du SNC. Le rôle de ces deux types de cellules diffère probablement dans le contexte neuro-inflammatoire caractérisant la sclérose en plaques (SP). Les macrophages accèdent alors au parenchyme et il devient difficile de les distinguer des microglies. Les expériences décrites dans la présente thèse ont été effectuées sur des microglies prélevées du SNC d'humains adultes et de cellules myéloïdes provenant du sang périphérique. Leurs réponses à différents stimuli ont été comparées. Les microglies et macrophages ont été exposés à la molécule FTY720, à des conditions expérimentales de polarisation ou à la myéline, une composante détruite dans la SP. La présence de l'acide ribonucléique codant pour les récepteurs de sphingosine-1-phosphate 1 à 5 (S1P1 à 5) a été utilisée comme indicateur du potentiel de réponse des cellules à l'agoniste FTY720. Il fut observé que les microglies, cellules dendritiques et macrophages dérivés de monocytes exprimaient le récepteur S1P1 à un niveau relativement plus élevé que les autres récepteurs (S1P2-5). En dépit de ce profil d'expression similaire, les voies de signalisation engagées par FTY720 ont différé entre les types de cellules. Les macrophages et cellules dendritiques traités avec FTY720 ont vu leur niveau de phospho-ERK baisser et leur phospho-MLC II augmenter tandis que les niveaux sont restés inchangés chez les microglies. FTY720 a mitigé la production d'interleukine (IL)-12p70 chez les cellules dendritiques et les macrophages (stimulées avec CD40L), mais pas chez les microglies (stimulées avec poly I:C). FTY720 a augmenté la production d'IL-10 chez les cellules dendritiques, mais est demeurée la même chez les autres cellules myéloïdes. Concernant les réponses à des conditions de polarisation, les macrophages peuvent être polarisés dans un spectre continu de phénotypes dont les extrêmes sont les phénotypes pro-inflammatoires « M1 » et anti-inflammatoires « M2 ». Ces phénotypes sont liés à la production de molécules inflammatoires et à l'activité phagocytaire des cellules. La fonction et le phénotype des microglies soumises aux conditions de polarisation pro-M1 et pro-M2 ont été comparés à celles des macrophages dérivés de monocytes sanguins périphériques dans des conditions semblables. Dans des conditions pro-M1, les microglies et les macrophages augmentaient leur expression des marqueurs M1 CCR7 et CD80. Les macrophages soumis à des conditions pro-M2 exprimaient les marqueurs C209, CD23, CD163 et CD206. Par contre, les microglies soumises à de telles conditions n'exprimaient que CD209. Les conditions pro-M1 ont amené les microglies et les macrophages à produire IL-12p40. Cependant, les microglies produisaient davantage d'IL-10 que les macrophages dans ces mêmes conditions. Les microglies stimulées par les signaux pro-M1 et pro-M2 produisaient des niveaux comparables de IL-10 tandis que les macrophages produisaient IL-10 avec des signaux M2 plus LPS. L'activité phagocytaire était plus élevée chez les microglies que chez les macrophages, peu importe la condition polarisante. Elle était cependant augmentée chez les deux types de cellules par des conditions pro-M2. Nos résultats mettent en évidence certaines propriétés distinctes des microglies et des cellules myéloïdes sanguines dans le contexte physiologique et face aux signaux provenant d'un SNC inflammé. Les donnés démontrant un comportement unique chez chacun des types de cellules myéloïdes en réponse à un agent thérapeutique approuvé pour la SP suggèrent que de cibler les populations myéloïdes peut avoir un impact. Favoriser une polarisation M2 chez les macrophages pourrait s'avérer une stratégie visant à contrôler l'inflammation du SNC.
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